JP6672343B2 - Cancer immunotherapy by disrupting PD-1 / PD-L1 signaling - Google Patents

Description

  This application claims the benefit of US Provisional Patent Application No. 61 / 647,442 filed May 15, 2012 and US Provisional Patent Application No. 61 / 790,747 filed March 15, 2013. , The contents of which are hereby incorporated by reference in their entirety.

  Throughout this application, various other publications are referred to in parentheses by author and date, or by patent or patent publication number. Full citations for these publications may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference, in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed in the present application. Included in the description. However, citation of a document should not be construed as an admission that such document is prior art to the present invention.

TECHNICAL FIELD The present invention relates to a method for immunotherapy of a cancer patient, comprising administering to the patient an antibody that disrupts the PD-1 / PD-L1 signaling pathway. Biomarkers can be used as part of a treatment to identify appropriate patients for immunotherapy and to predict the efficacy of anti-PD-1 treatment.

BACKGROUND OF THE INVENTION Human cancers have numerous genes and epigenetic alterations that produce new antigens that are potentially recognizable by the immune system (Sjoblom et al., 2006). The adaptive immune system, composed of T and B lymphocytes, has a broad capacity to respond to a variety of tumor antigens and the potential for strong anti-cancer with excellent specificity. In addition, the immune system has demonstrated considerable plasticity and memory components. Successful utilization of all these properties of the adaptive immune system makes immunotherapy unique in all modes of cancer treatment. However, although an endogenous immune response to cancer is observed in preclinical models and patients, this response is not effective and established cancers are considered "self" by the immune system and are tolerated. This state of tolerance allows tumors to take advantage of several different mechanisms and actively destroy anti-tumor immunity. These mechanisms down regulate the strength of the adaptive immune response by dysfunctional T-cell signaling (Mizoguchi et al., 1992), suppressor regulatory cells (Facciabene et al., 2012), and tumors to avoid immune destruction. And co-opting of endogenous "immune checkpoints" to help protect normal tissues from collateral damage (Topalian et al., 2011; Mellman et al., 2011).

  Until recently, cancer immunotherapy has made considerable effort in approaches to enhance anti-tumor immune responses by adoptive transfer of activated effector cells, immunization against relevant antigens, or provision of non-specific immunostimulants, such as cytokines. Was concentrated. However, in the last decade, a thorough effort to develop specific immune checkpoint pathway inhibitors has resulted in cytotoxic T-lymphocyte antigen-4 (CTLA-4) for the treatment of patients with advanced melanoma. An antibody (Ab) that binds and inhibits the development of ipilimumab (YERVOY®) (Hodi et al., 2010) and, as described herein, inhibits the inhibitory PD-1 pathway. It has begun to provide a new immunotherapeutic approach to treating cancer, including the development of blocking Abs.

  Programmed death-1 (PD-1) is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, including CD28, CTLA-4, ICOS, PD-1 and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, and programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2) have been identified in antigen-presenting cells and many human cancers. Expressed and shown to bind to PD-1 and downregulate T cell activation and cytokine secretion (Freeman et al., 2000; Latchman et al., 2001). Unlike CTLA-4, PD-1 is an immunosuppressive PD-L1 (B7-H1) and PD-L2 (B7-DC) in which activated T-cells are expressed by tumors and / or stromal cells. It functions primarily in peripheral tissues where it can encounter ligands (Flies et al., 2011; Topalian et al., 2012a). Inhibition of the PD-1 / PD-L1 interaction mediates potent antitumor activity in preclinical models (U.S. Pat. Nos. 8,008,449 and 7,943,743) and PD for treating cancer The use of Ab inhibitors of the -1 / PD-L1 interaction has entered a clinical trial (Brahmer et al., 2010; Topalian et al., 2012b; Brahmer et al., 2012; Flies et al., 2011; Pardoll, 2012; Hamid and Carvajal, 2013). .

  The promise of a new field of personalized medicine is that advances in pharmacogenomics will tailor treatment to defined subpopulations, and ultimately individual patients, to enhance efficacy and minimize side effects Is to be used incrementally. Recent successes include, for example, imatinib mesylate (GLEEVEC®), a protein tyrosine kinase inhibitor that inhibits the bcr-abl tyrosine kinase to treat Philadelphia chromosome positive chronic myeloid leukemia (CML); Crizotinib (XALKORI®) treating 5% of patients with late stage non-small cell lung cancer expressing the anaplastic lymphoma kinase (ALK) gene; and the mutant B- expressed in about half of melanoma tumors Includes the development of Vemurafenib (ZELBORAF®), an inhibitor of the RAF protein (V600E-BRAF). However, unlike the clinical development of small molecule agents that target discrete activating mutations found in selected cancer populations, certain challenges in cancer immunotherapy allow for patient selection and guide treatment management Therefore, it is said that the identification of mechanism-based predictive biomarkers. Described herein are advances in confirming PD-L1 expression as a biomarker to screen patients for anti-PD-1 immunotherapy.

SUMMARY OF THE INVENTION The present specification is a method for immunotherapy of a subject suffering from cancer, comprising a therapeutically effective amount that disrupts, reduces, or suppresses signaling from an inhibitory immunoregulator. Provided to a subject. In a preferred embodiment, the drug is an Ab. In another preferred embodiment, the inhibitory immunoregulator is a component of the PD-1 / PD-L1 signaling pathway. In a further preferred embodiment, the Ab disrupts the interaction between PD-1 and PD-L1. In one embodiment, the Ab is an anti-PD-1 Ab of the invention or an anti-PD-L1 Ab of the invention. In a preferred embodiment, the anti-PD-1 Ab of the invention is nivolumab (BMS-936558) and the anti-PD-L1 Ab of the invention is BMS-936559. In one embodiment, the subject has been pre-treated for cancer. In other embodiments, the cancer is an advanced, metastatic and / or refractory cancer. In a preferred embodiment, administration of the Ab or antigen-binding portion thereof to a subject induces a sustained clinical response in the subject.

  The application also provides (a) (i) optionally, a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells, (Ii) assessing the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample; and (iii) determining the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample in advance. Selecting a subject that is a suitable candidate for immunotherapy, including selecting the subject as a suitable candidate based on the assessment that the determined threshold level is exceeded; and (b) PD-1 / PD Administering to a selected subject a composition comprising a therapeutically effective amount of an agent that disrupts signaling from the L1 pathway, such as an anti-PD-1 or anti-PD-L1 Ab. Provides methods for immunotherapy of a subject suffering from cancer.

  The present application provides (a) (i) optionally a test tissue sample obtained from a patient having a tissue cancer, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; ii) assessing the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample; and (iii) predetermining the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample. Agents that disrupt signaling from the PD-1 / PD-L1 pathway, including selecting subjects as unsuitable for immunotherapy, based on the assessment that they are below a threshold level determined, e.g., anti-PD- Selecting subjects unsuitable for immunotherapy with 1 or anti-PD-L1 Ab; and (b) other than agents that disrupt signaling from the PD-1 / PD-L1 pathway Comprising administering to a subject a selected standard therapeutic treatment further provides a method for the treatment of a subject suffering from cancer.

  In addition, the present application provides (a) optionally, a test tissue sample obtained from a patient having a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; A) assaying the test tissue sample to determine the percentage of cells expressing PD-L1 on the cell surface; (c) determining the percentage of cells expressing PD-L1 on the cell surface at a predetermined threshold. PD-1 / PD-L1 pathway, including comparing to percentages; and (d) selecting patients for immunotherapy based on the assessment that PD-L1 is expressed in cells of a test tissue sample. Methods for selecting cancer patients for immunotherapy with agents that disrupt signal transduction from, for example, anti-PD-1 or anti-PD-L1 Ab.

  The present application provides (a) optionally, a test tissue sample obtained from a patient having a tissue cancer, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (b) cell surface Assaying a test tissue sample to determine the percentage of cells expressing PD-L1 above; (c) comparing the percentage of cells expressing PD-L1 on the cell surface to a predetermined threshold. And (d) agents that disrupt signaling from the PD-1 / PD-L1 pathway, such as anti-PD-1 or for treating cancer patients, including predicting the therapeutic effect of the agent. A method for predicting the therapeutic effect of an anti-PD-L1 Ab, wherein the agent treats a patient when the percentage of cells expressing PD-L1 on the cell surface exceeds a threshold percentage. Predicted to be effective, when the percentage of cells expressing PD-L1 on the cell surface is less than the threshold percentage, the drug is not expected to be effective in treating patients, further provides methods.

  The present description also provides (a) optionally, a test tissue sample obtained from a patient having a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (b) Assaying the test tissue sample to determine the percentage of cells expressing PD-L1 on the cell surface; (c) determining the percentage of cells expressing PD-L1 on the cell surface by a predetermined threshold percentage And (d) determining an immunotherapeutic regimen comprising the agent based on a determination that the percentage of cells expressing PD-L1 on the cell surface exceeds a predetermined threshold percentage. Immunotherapeutic agents comprising agents that disrupt signaling from the PD-1 / PD-L1 pathway, such as anti-PD-1 or anti-PD-L1 Ab, for treating cancer patients It provides a method for determining the member.

  In one aspect of the methods described herein, the test tissue sample is a formalin fixed paraffin embedded (FFPE) sample. In one other embodiment, assessing the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample is accomplished by immunohistochemical (IHC) staining of the FFPE sample. In a preferred embodiment, mAb 28-8 or 5H1 is used in an automated IHC assay to bind PD-L1 on the surface of cells in a test tissue sample. In a preferred embodiment of any of the methods described herein, the cancer is melanoma (MEL), renal cell carcinoma (RCC), squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colon Rectal cancer (CRC), castration resistant prostate cancer (CRPC), hepatocellular carcinoma (HCC), squamous cell carcinoma of the head and neck, carcinoma of the esophagus, ovary, gastrointestinal tract and breast, or hematologic malignancy, eg, multiple bone marrow Tumors, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, and chronic myeloid leukemia.

  The invention further provides a mAb or antigen-binding portion thereof that specifically binds to a human PD-L1 antigen expressed on the cell surface in a FFPE tissue sample. In a preferred embodiment, the mAb or antigen binding portion thereof does not bind to cytoplasmic PD-L1 antigen in FFPE tissue samples. In other preferred embodiments, the monoclonal Ab (mAb) is a rabbit mAb designated 28-8, 28-1, 28-12, 29-8 or 20-12, or a mouse mAb designated 5H1.

  Other features and advantages of the invention will be apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, including scientific articles, newspaper reports, GenBank registrations, patents and patent applications, cited throughout this application are expressly incorporated herein by reference.

Cross-competition between 5C4 and other HuMab anti-PD-1 mAbs for binding to human PD-1 (hPD-1) expressed on CHO cells. A, 5C4 Fab fragment substantially blocked the binding of mAb 5C4 itself, as well as 2D3 and 7D3; B, 5C4 Fab fragment substantially blocked the binding of mAb 4H1; C, 5C4 mAb , MAb 17D8 binding was substantially blocked. Cross-competition between 5C4 and other HuMab anti-PD-1 mAbs for binding to human PD-1 (hPD-1) expressed on CHO cells. A, 5C4 Fab fragment substantially blocked the binding of mAb 5C4 itself, as well as 2D3 and 7D3; B, 5C4 Fab fragment substantially blocked the binding of mAb 4H1; C, 5C4 mAb , MAb 17D8 binding was substantially blocked. Cross-competition between 5C4 and other HuMab anti-PD-1 mAbs for binding to human PD-1 (hPD-1) expressed on CHO cells. A, 5C4 Fab fragment substantially blocked the binding of mAb 5C4 itself, as well as 2D3 and 7D3; B, 5C4 Fab fragment substantially blocked the binding of mAb 4H1; C, 5C4 mAb , MAb 17D8 binding was substantially blocked.

Cross-competition of human anti-hPD-L1 mAb conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4 and significantly blocked by itself; B, binding of labeled 3G10 was measured for each tested anti-PD except 10H10. C, the binding of labeled 10A5 was significantly blocked by each tested anti-PD-L1 Ab except 10H10; D, the binding of labeled 11E6 was 10H10. And E, the binding of labeled 12A4 was significantly blocked by each tested anti-PD-L1 Ab except 10H10. And F, binding of labeled 13G4 to each tested anti-PD-L1 Ab except 10H10 Significantly blocked. Cross-competition of human anti-hPD-L1 mAb conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4 and significantly blocked by itself; B, binding of labeled 3G10 was measured for each tested anti-PD except 10H10. C, the binding of labeled 10A5 was significantly blocked by each tested anti-PD-L1 Ab except 10H10; D, the binding of labeled 11E6 was 10H10. And E, the binding of labeled 12A4 was significantly blocked by each tested anti-PD-L1 Ab except 10H10. And F, binding of labeled 13G4 to each tested anti-PD-L1 Ab except 10H10 Significantly blocked. Cross-competition of human anti-hPD-L1 mAb conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4 and significantly blocked by itself; B, binding of labeled 3G10 was measured for each tested anti-PD except 10H10. C, the binding of labeled 10A5 was significantly blocked by each tested anti-PD-L1 Ab except 10H10; D, the binding of labeled 11E6 was 10H10. And E, the binding of labeled 12A4 was significantly blocked by each tested anti-PD-L1 Ab except 10H10. And F, binding of labeled 13G4 to each tested anti-PD-L1 Ab except 10H10 Significantly blocked. Cross-competition of human anti-hPD-L1 mAb conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4 and significantly blocked by itself; B, binding of labeled 3G10 was measured for each tested anti-PD except 10H10. C, the binding of labeled 10A5 was significantly blocked by each tested anti-PD-L1 Ab except 10H10; D, the binding of labeled 11E6 was 10H10. And E, the binding of labeled 12A4 was significantly blocked by each tested anti-PD-L1 Ab except 10H10. And F, binding of labeled 13G4 to each tested anti-PD-L1 Ab except 10H10 Significantly blocked. Cross-competition of human anti-hPD-L1 mAb conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4 and significantly blocked by itself; B, binding of labeled 3G10 was measured for each tested anti-PD except 10H10. C, the binding of labeled 10A5 was significantly blocked by each tested anti-PD-L1 Ab except 10H10; D, the binding of labeled 11E6 was 10H10. And E, the binding of labeled 12A4 was significantly blocked by each tested anti-PD-L1 Ab except 10H10. And F, binding of labeled 13G4 to each tested anti-PD-L1 Ab except 10H10 Significantly blocked. Cross-competition of human anti-hPD-L1 mAb conjugated to FITC for binding to human PDF-L1 (hPD-L1) expressed on CHO cells. A, binding of labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4 and significantly blocked by itself; B, binding of labeled 3G10 was measured for each tested anti-PD except 10H10. C, the binding of labeled 10A5 was significantly blocked by each tested anti-PD-L1 Ab except 10H10; D, the binding of labeled 11E6 was 10H10. And E, the binding of labeled 12A4 was significantly blocked by each tested anti-PD-L1 Ab except 10H10. And F, binding of labeled 13G4 to each tested anti-PD-L1 Ab except 10H10 Significantly blocked.

Cross-competitive inhibition of binding of biotinylated mAb 12A4 to ES-2 cells by human anti-hPD-L1 mAb. The fluorescence of the bound biotin-12A4 is plotted against the concentration of unlabeled hPD-L1 HuMab.

Spider plot showing activity of anti-PD-1 mAb in patients with refractory melanoma (MEL). A typical plot of the change in tumor burden with time is shown in the sum of the longest diameters of target lesions in 27 MEL patients treated with 5C4 at a dose of 1.0 mg / kg compared to baseline. Prove the time course of change. In the majority of patients who achieved an objective response (OR), the response was persistent and evident by the end of cycle 3 of treatment (June) (dashed vertical line). Tumor regression, for example, a prolonged decrease in tumor burden in the presence of new lesions, followed a conventional and "immune-related" pattern of response.

Activity of anti-PD-1 mAb in patients with metastatic RCC. FIG. 4 illustrates partial regeneration of metastatic RCC in a 57 year old patient treated with 5C4 at 1 mg / kg. The patient had previously undergone curative surgery and developed progressive disease after receiving sunitinib, temsirolimus, sorafenib and pazopanib. Arrows indicate regression of recurrent tumors in the surgical field.

Activity of anti-PD-1 mAb in patients with metastatic MEL. A complete response of metastatic MEL is illustrated in a 62 year old patient treated with 5C4 at 3 mg / kg associated with vitiligo. (I) Pre-treatment CT scan, inguinal lymph node metastasis (arrow); (ii) 13 months after treatment. Numerous metastases in the subcutaneous tissue and retroperitoneum were also completely regressed (not shown). Vitiligo developed after 6 months of treatment; photos were taken in September under visible light (iii) and ultraviolet light (iv). Skin biopsy by immunohistochemistry for microphthalmia-associated transcription factor (MITF) is partially or completely affected by melanocytes (arrows) at the epidermal-dermal border in normal skin (v), and vitiligo (Vi) or absent (vii) melanocytes in the skin.

Activity of anti-PD-1 mAb in patients with metastatic NSCLC. A partial response is described in a patient with metastatic NSCLC (non-squamous epithelial tissue) treated with 5C4 at 10 mg / kg. Arrows indicate early progression in lung lesions, followed by regression ("immune-related" pattern of response).

Correlation between tumor PD-L1 expression and anti-PD-1 clinical response. Pretreated tumor cell surface expression of PD-L1 correlates with OR for PD-1 blockade as determined by IHC on formalin fixed paraffin embedded specimens. Study 42 subjects with advanced cancers including melanoma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma and castration-resistant prostate cancer (n = 18, 10, 7, 5, and 2, respectively) did. A, There was a significant correlation between objective clinical response and tumor cell surface PD-L1 expression. Patients with PD-L1 negative tumor did not experience OR. B, Examples of IHC analysis with anti-PD-L1 mAb 5H1 are shown in lymph node metastasis of melanoma (top), nephrectomized specimen of renal cell carcinoma (middle), and brain metastasis of lung adenocarcinoma (bottom). It is. All are 400X original magnification. Arrows indicate one of a number of tumor cells in each specimen with surface membrane staining for PD-L1. Asterisks indicate normal glomeruli in nephrectomized specimens that are negative for PD-L1 staining. Correlation between tumor PD-L1 expression and anti-PD-1 clinical response. Pretreated tumor cell surface expression of PD-L1 correlates with OR for PD-1 blockade as determined by IHC on formalin fixed paraffin embedded specimens. Study 42 subjects with advanced cancers including melanoma, non-small cell lung cancer, colorectal cancer, renal cell carcinoma and castration-resistant prostate cancer (n = 18, 10, 7, 5, and 2, respectively) did. A, There was a significant correlation between objective clinical response and tumor cell surface PD-L1 expression. Patients with PD-L1 negative tumor did not experience OR. B, Examples of IHC analysis with anti-PD-L1 mAb 5H1 are shown in lymph node metastasis of melanoma (top), nephrectomized specimen of renal cell carcinoma (middle), and brain metastasis of lung adenocarcinoma (bottom). It is. All are 400X original magnification. Arrows indicate one of a number of tumor cells in each specimen with surface membrane staining for PD-L1. Asterisks indicate normal glomeruli in nephrectomized specimens that are negative for PD-L1 staining.

Graphical comparison of mAb 28-8 and 5H1 binding to PD-L1 antigen in tumor tissue by histoscore analysis. Rabbit mAb 28-8 showed a higher histo-score in 7 out of 10 samples tested.

Spider plot showing activity of anti-PD-L1 mAb in patients with refractory MEL and NSCLC. Typical plots show patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and NSCLC treated with 10 mg / kg (D) Figure 9 demonstrates the time course of target lesion tumor burden in patients with a. In the majority of patients who achieved OR, the response was persistent and evident by the end of cycle 2 of treatment (March), regardless of dose or tumor type. Tumor regression followed a conventional and "immune-related" pattern of response. Spider plot showing activity of anti-PD-L1 mAb in patients with refractory MEL and NSCLC. Typical plots show patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and NSCLC treated with 10 mg / kg (D) Figure 9 demonstrates the time course of target lesion tumor burden in patients with a. In the majority of patients who achieved OR, the response was persistent and evident by the end of cycle 2 of treatment (March), regardless of dose or tumor type. Tumor regression followed a conventional and "immune-related" pattern of response. Spider plot showing activity of anti-PD-L1 mAb in patients with refractory MEL and NSCLC. Typical plots show patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and NSCLC treated with 10 mg / kg (D) Figure 9 demonstrates the time course of target lesion tumor burden in patients with a. In the majority of patients who achieved OR, the response was persistent and evident by the end of cycle 2 of treatment (March), regardless of dose or tumor type. Tumor regression followed a conventional and "immune-related" pattern of response. Spider plot showing activity of anti-PD-L1 mAb in patients with refractory MEL and NSCLC. Typical plots show patients with MEL treated with BMS-936559 at doses of 1 (A), 3 (B), 10 mg / kg (C), and NSCLC treated with 10 mg / kg (D) Figure 9 demonstrates the time course of target lesion tumor burden in patients with a. In the majority of patients who achieved OR, the response was persistent and evident by the end of cycle 2 of treatment (March), regardless of dose or tumor type. Tumor regression followed a conventional and "immune-related" pattern of response.

Complete response in a patient with melanoma treated with BMS-936559 at 3 mg / kg. Circles indicate an initial increase in pulmonary nodule size at 6 weeks and March, followed by complete regression ("immune-related" pattern of response) at October.

Complete response in a patient with melanoma treated with BMS-936559 at 1 mg / kg. The patient developed a solitary brain metastasis 3 months after the start of treatment successfully treated with stereotactic radiotherapy. Partial response in abdominal disease (circled) was shown in August, with no signs of disease at 15 months.

Partial response in patients with NSCLC (non-squamous epithelium) treated with BMS-936559 at 10 mg / kg. 2 shows the response in diseases in the right lung pleura and liver.

Clinical activity of MEL patients receiving a concurrent regimen of nivolumab and ipilimumab. A, A typical spider plot is the baseline in tumor burden measured as the sum of the product of the vertical diameters of all target lesions in patients receiving a concurrent regimen of 1 mg / kg nivolumab + 3 mg / kg ipilimumab at MTD It shows the change from. The triangle indicates the first occurrence of a new lesion. B, Typical waterfall plot shows the maximum response rate at baseline target lesion in patients receiving a concurrent regimen. Clinical activity of MEL patients receiving a concurrent regimen of nivolumab and ipilimumab. A, A typical spider plot is the baseline in tumor burden measured as the sum of the product of the vertical diameters of all target lesions in patients receiving a concurrent regimen of 1 mg / kg nivolumab + 3 mg / kg ipilimumab at MTD It shows the change from. The triangle indicates the first occurrence of a new lesion. B, Typical waterfall plot shows the maximum response rate at baseline target lesion in patients receiving a concurrent regimen.

Tumor regression in a 52-year-old MEL patient receiving a concurrent regimen of 1 mg / kg nivolumab + 3 mg / kg ipilimumab. The patient had extensive neck, mediastinal, axillary, abdominal and pelvic lymphadenopathy, bilateral pulmonary nodules, small intestinal metastases, peritoneal implants and extensive subcutaneous nodules. Baseline lactate dehydrogenase (LDH) was at the upper limit of 2.25x normal, hemoglobin was 9.7 g / dL, and symptoms included nausea and vomiting. Within 4 weeks of treatment, LDH normalized, symptoms improved (increased appetite, decreased vomiting) and skin damage regressed. At the 12-week scan, there was a significant reduction in all areas of the disease. Arrows indicate the location of metastatic disease.

. Clinical activity of MEL patients receiving various concurrent regimens of nivolumab and ipilimumab. A typical spider-like plot shows a concurrent regimen of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab (A), 3 mg / kg nivolumab + 1 mg / kg ipilimumab (B), or 3 mg / kg nivolumab + 3 mg / kg ipilimumab (C). Figure 3 shows the change from baseline in tumor burden, measured as the sum of the products of the vertical diameters of all target lesions, in patients who received it. The triangle indicates the first occurrence of a new lesion. . Clinical activity of MEL patients receiving various concurrent regimens of nivolumab and ipilimumab. A typical spider-like plot shows a concurrent regimen of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab (A), 3 mg / kg nivolumab + 1 mg / kg ipilimumab (B), or 3 mg / kg nivolumab + 3 mg / kg ipilimumab (C). Figure 3 shows the change from baseline in tumor burden, measured as the sum of the products of the vertical diameters of all target lesions, in patients who received it. The triangle indicates the first occurrence of a new lesion. . Clinical activity of MEL patients receiving various concurrent regimens of nivolumab and ipilimumab. A typical spider-like plot shows a concurrent regimen of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab (A), 3 mg / kg nivolumab + 1 mg / kg ipilimumab (B), or 3 mg / kg nivolumab + 3 mg / kg ipilimumab (C). Figure 3 shows the change from baseline in tumor burden, measured as the sum of the products of the vertical diameters of all target lesions, in patients who received it. The triangle indicates the first occurrence of a new lesion.

Tumor regression of a 61-year-old MEL patient receiving a concurrent regimen of 0.3 mg / kg nivolumab + 3 mg / kg ipilimumab. The patient showed gastric and mesenteric metastases with stage IV (M1c) MEL of unknown primary site. After a recent transfusion, lactate dehydrogenase was 225 and hemoglobin was 9.6 g / dL. Twelve weeks after the start of the study treatment, a CT scan showed a 86% reduction in large disease burden.

Clinical activity of MEL patients receiving a continuous regimen of nivolumab and ipilimumab. A typical spider plot is the sum of the product of the vertical diameters of all target lesions in patients receiving a continuous regimen of 1 mg / kg nivolumab (A) or 3 mg / kg nivolumab (B) before and after ipilimumab treatment. Figure 3 shows the change from baseline in measured tumor burden. The triangle indicates the first occurrence of a new lesion. C, Typical waterfall plot shows maximum response rate at baseline target lesion in patients receiving a continuous regimen. "*" Indicates patients with radiation progression on previous ipilimumab treatment. Clinical activity of MEL patients receiving a continuous regimen of nivolumab and ipilimumab. A typical spider plot is the sum of the product of the vertical diameters of all target lesions in patients receiving a continuous regimen of 1 mg / kg nivolumab (A) or 3 mg / kg nivolumab (B) before and after ipilimumab treatment. Figure 3 shows the change from baseline in measured tumor burden. The triangle indicates the first occurrence of a new lesion. C, Typical waterfall plot shows maximum response rate at baseline target lesion in patients receiving a continuous regimen. "*" Indicates patients with radiation progression on previous ipilimumab treatment. Clinical activity of MEL patients receiving a continuous regimen of nivolumab and ipilimumab. A typical spider plot is the sum of the product of the vertical diameters of all target lesions in patients receiving a continuous regimen of 1 mg / kg nivolumab (A) or 3 mg / kg nivolumab (B) before and after ipilimumab treatment. Figure 3 shows the change from baseline in measured tumor burden. The triangle indicates the first occurrence of a new lesion. C, Typical waterfall plot shows maximum response rate at baseline target lesion in patients receiving a continuous regimen. "*" Indicates patients with radiation progression on previous ipilimumab treatment.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to compositions comprising a therapeutically effective amount of a compound or agent that enhances an endogenous immune response that stimulates activation of an endogenous response or inhibits suppression of an endogenous response. For immunotherapy of a subject suffering from a disease, for example, cancer or an infectious disease, comprising administering to a subject. More specifically, this application involves administering to a subject a therapeutically effective amount of an agent that disrupts or inhibits signaling from an inhibitory immune regulator, eg, an Ab or antigen-binding portion thereof. There is provided a method for enhancing an endogenous immune response in a subject afflicted with, thereby treating a patient. In one embodiment, the inhibitory immune regulator is a component of the PD-1 / PD-L1 signaling pathway. Accordingly, one aspect of the present invention relates to a method of treating cancer comprising administering to a subject a therapeutically effective amount of an Ab or antigen-binding portion thereof that disrupts the interaction between the PD-1 receptor and its ligand, PD-L1. A method for immunotherapy of a subject suffering from is provided. In one preferred embodiment, the Ab or antigen binding portion thereof specifically binds to PD-1. In another preferred embodiment, the Ab or antigen-binding portion thereof specifically binds to PD-L1. One embodiment involves the use of an anti-PD-1 Ab in combination with another anti-cancer agent, preferably an anti-CTLA-4 Ab, to treat cancer. In one other embodiment, the subject measures the surface expression of PD-L1 in a test tissue sample obtained from a patient with cancer of the tissue, eg, expresses PD-L1 on the cell surface in the test tissue sample. Determining the percentage of cells to perform, and selecting patients for immunotherapy based on the assessment that PD-L1 is expressed on the surface of cells in a test tissue sample. Selected as appropriate.

Terminology In order that the specification may be more readily understood, certain terms are first defined. As used herein, each of the following terms has the meaning set forth below, unless expressly provided otherwise herein. Further definitions are set forth in this application.

  "Administration" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Preferred routes of administration for the Abs of the present invention include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal or other parenteral routes of administration, for example by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, Intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, and intrasternal injection and infusion , As well as in vivo electroporation. Alternatively, the Abs of the invention can be administered, for example, intranasally, orally, intravaginally, rectally, sublingually, or topically, by non-parenteral routes, such as topical, epidermal or mucosal routes of administration. Can be administered. Administration can also be performed, for example, once, multiple times and / or one or more times over an extended period.

  As used herein, an "adverse event" (AE) refers to any undesired and generally unintended or undesired signs (including abnormal laboratory findings), symptoms, symptoms, Or is a disease. For example, an adverse event can be associated with activation of the immune system in response to treatment or expansion of immune system cells (eg, T cells). A medical treatment can have one or more associated AEs, and each AE can have the same or different levels of severity. Reference to a method that can “change the adverse event” refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of different treatment regimens.

"Antibodies" (Abs) include, but are not limited to, glycoprotein immunoglobulins that specifically bind to an antigen, and have at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Or an antigen binding portion thereof. Each H chain includes a heavy chain variable region (abbreviated herein as V _H) and a heavy chain constant region. The heavy chain constant region includes three constant _domains, C _{H1, C H2} and _{C H3.} Each light chain includes a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region comprises one constant domain, a C _L. The _VH and _VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which incorporate more conserved regions, called framework regions (FR). . Each _VH and _VL contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the Ab may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

Antibodies typically bind specifically to their cognate antigen with high affinity, as reflected by a dissociation constant (K _D ) of 10 ⁻⁵ to 10 ⁻¹¹ M ⁻¹ or less. About ¹⁰ -4 ^{M -1} or more optional a _{K D,} generally considered to indicate non-specific binding. As used herein, an Ab that “specifically binds” to an antigen is 10 ⁻⁷ M or less, preferably 10 ⁻⁸ M or less, even more preferably 5 × 10 ⁻⁹ M or less, and more preferably 10 ⁻¹⁰ M or less. with high affinity, which means that from ^{-8 M} having a 10 ^{-10 M} or less for K _D, that bind to the antigen and substantially identical to an antigen, but exhibit Ab does not bind to the antigen that is not associated with high affinity. An antigen exhibits a high degree of sequence identity with a given antigen, eg, at least 80%, at least 90%, preferably at least 95%, more preferably at least 97%, or even more preferably, with the sequence of the given antigen. "Substantially identical" to a given antigen when it exhibits at least 99% sequence identity. As an example, Abs that specifically bind to human PD-1 may also be cross-reactive with PD-1 antigens from certain primate species, but PD-1 antigens from certain rodent species Or, it cannot cross-react with an antigen other than PD-1, for example, a human PD-L1 antigen.

  Immunoglobulins may be from any of the commonly known isotypes, including but not limited to IgA, secreted IgA, IgG and IgM. IgG subclasses are also well known to those of skill in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. “Isotype” indicates the Ab class or subclass (eg, IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, by way of example, both natural and non-natural Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Except where expressly stated, unless the context indicates otherwise, the term "antibody" also includes any antigen binding fragment or portion of the immunoglobulin, including monovalent and Includes bivalent fragments or moieties, and single-chain Abs.

  An “isolated antibody” refers to an Ab that is substantially free of other Abs having different antigenic specificities (eg, an isolated Ab that specifically binds to PD-1 is other than PD-1 Ab that specifically binds to the antigen is substantially not included). However, isolated Abs that specifically bind to PD-1 may have cross-reactivity to other antigens, such as PD-1 molecules from different species. Further, an isolated Ab may be substantially free of other cellular materials and / or chemicals. By comparison, an “isolated” nucleic acid is a nucleic acid composition of a substance that is significantly different from the nucleic acid as it exists in nature, ie, has unique chemical identity, properties, and utility. For example, isolated DNA, unlike natural DNA, is a free-standing portion of natural DNA, not a larger structural complex found in nature, an integral part of the chromosome. Furthermore, the isolated DNA can be used in applications or methods where natural genomic DNA is unsuitable, unlike natural genomic DNA, for example, for diagnosing disease or assessing the efficacy of treatment, among others. Can be commonly used as PCR primers or hybridization probes to measure gene expression, and to detect biomarker genes or mutations. The isolated nucleic acid is purified to be substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or proteins, using standard techniques well known in the art. obtain. Examples of isolated nucleic acids include genomic DNA, PCR-amplified DNA, cDNA and RNA fragments.

  The term “monoclonal antibody” (“mAb”) refers to a preparation of Ab molecules of a single molecular composition, ie, Ab molecules that are essentially identical in primary sequence, and that have a single binding specificity for a particular epitope Indicate sex and affinity. mAbs are examples of isolated Abs. MAbs can be produced by hybridomas, recombinant, transgenic, or other techniques known to those of skill in the art.

  A "human" antibody (HuMAb) refers to an Ab that has a variable region in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Further, when the Ab comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human Abs of the present invention include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). May be included. However, as used herein, the term "human antibody" includes an Ab in which CDR sequences from another mammalian species, for example, a mouse germline, are grafted onto a human framework sequence. Not intended. The terms "human" Ab and "fully human" Ab are used interchangeably.

  A “humanized” antibody refers to an Ab in which some, most, or all amino acids outside the CDR domains of a non-human Ab have been replaced with the corresponding amino acids from human immunoglobulin. In one embodiment of the humanized form of the Ab, some, most or all amino acids outside the CDR domains have been replaced with amino acids from a human immunoglobulin, but some within one or more CDR regions, Most or all amino acids are unchanged. Some additions, deletions, insertions, substitutions or modifications of amino acids are allowed, as long as the Ab does not abolish its ability to bind to a particular antigen. A "humanized" Ab retains the same antigen specificity as the original Ab.

  A “chimeric antibody” is an Ab in which the variable region is from one species and the constant region is from another species, for example, an Ab in which the variable region is from a mouse Ab and the constant region is from a human Ab Is shown.

  An “antigen-binding portion” of an Ab (also referred to as an “antigen-binding fragment”) refers to one or more fragments of an Ab that retains the ability to specifically bind to the antigen bound by the complete Ab.

  "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation Division and proliferation cause the formation of malignant tumors that can invade adjacent tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream.

  An "immune response" refers to the selective targeting, binding to invasive pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, to normal human cells or tissues. Cells of the immune system (eg, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendrites, causing damage, destruction and / or removal from the vertebrate body 9 shows the effects of soluble macromolecules (including Abs, cytokines and complement) produced by these cells in cells and neutrophils) and liver.

  "Immune regulator" refers to a substance, agent, signal transduction pathway or component thereof that modulates the immune response. "Control," "modification," or "modulation" of an immune response refers to any change in cells of the immune system or the activity of such cells. Such control may include an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or a promotion or suppression of the immune system that may be manifested by any other change that can occur within the immune system. Including. Both inhibitory and stimulatory immune regulators have been identified, some of which may have enhanced function in the cancer microenvironment.

  The term “immunotherapy” refers to the treatment of a subject having, or at risk of having, or having a relapse of a disease by a method comprising inducing, enhancing, suppressing or modifying an immune response. Indicates treatment. "Treatment" or "therapy" in a subject reverses, alleviates, ameliorates, inhibits, delays or prevents the onset, progression, development, severity or relapse of symptoms, complications, conditions or biochemical signs associated with the disease It indicates any type of intervention or process performed on a subject with an objective, or administration of an active agent to the subject.

  “Enhancing the endogenous immune response” means increasing the effectiveness or potency of an immune response present in a subject. This increase in efficacy and potency can be achieved, for example, by overcoming a mechanism that suppresses the endogenous host immune response or by stimulating a mechanism that enhances the endogenous host immune response.

  A “predetermined threshold” for cell surface PD-L1 expression is defined as the number of cells in a test tissue sample containing tumor cells and tumor-infiltrating inflammatory cells where the sample is scored positive for cell surface PD-L1 expression. Shows the ratio of In cell surface expression assayed by IHC with mAb 28-8, the predetermined threshold for cells expressing PD-L1 on the cell surface ranges from at least about 0.01% to at least about 20% of the total number of cells. It is. In a preferred embodiment, the predetermined threshold for cells expressing PD-L1 on the cell surface ranges from at least about 0.1% to at least about 10% of the total number of cells. More preferably, the predetermined threshold is at least about 5%. Even more preferably, the predetermined threshold is at least about 1%, or in the range of 1-5%.

  "Programmed death-1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is predominantly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein refers to human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and at least one common epitope with hPD-1 And analogs having The complete hPD-1 sequence can be found under GenBank accession number U64863.

  "Programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 that, when bound to PD-1, downregulates T cell activation and cytokine secretion (others). Is PD-L2). As used herein, the term “PD-L1” refers to human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and at least one common epitope with hPD-L1 And analogs having The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

  "Signal transduction pathway" or "signal transduction pathway" refers to a biochemical relationship between various signal transducing molecules that plays a role in transmitting signals from one part of a cell to another part of a cell. "Cell surface receptors" include, for example, molecules and complexes of molecules that are located on the surface of a cell, receive signals, and can transmit such signals through the cell membrane of the cell. Examples of cell surface receptors of the present invention include PD-1 located on the surface of activated T cells, activated B cells and bone marrow cells and transducing signals that cause a decrease in tumor infiltrating lymphocytes and a decrease in T cell proliferation. Is a receptor. An "inhibitor" of signal transduction refers to a compound or agent that antagonizes or reduces the initiation, acceptance, or transduction of a signal that is transduced by a component of a signaling pathway, such as a receptor or its ligand, in a stimulatory or inhibitory manner. .

  "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, cats, rabbits and ferrets, rodents such as mice, rats and guinea pigs, birds such as, for example, Includes chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a mammal, eg, a non-human primate, sheep, dog, cat, rabbit, ferret, or rodent. In a more preferred embodiment, the subject is a human. The terms "subject", "patient" and "individual" are used interchangeably herein.

  A “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent, eg, an Ab of the present invention, when used alone or in combination with another therapeutic agent, decreases the severity of disease symptoms, Any amount of drug that protects the subject against the onset of the disease or promotes regression of the disease, as evidenced by an increase in the frequency and duration of the asymptomatic period, or prevention of injury or disability due to disease pain. It is. The ability of a therapeutic agent to promote disease regression can be determined, for example, in human subjects in clinical trials, in animal model systems that predict efficacy in humans, using various methods known to those of skill in the art, or It can be assessed by assaying the activity of the drug in an in vitro assay.

  As an example, the anticancer agent promotes regression of the cancer in the subject. In a preferred embodiment, the therapeutically effective amount of the drug promotes regression of the cancer to the point of eliminating the cancer. "Promote cancer regression" means that administration of an effective amount of the drug, alone or in combination with an anti-tumor agent, reduces tumor growth or tumor size, necrosis of the tumor, or reduces the severity of at least one disease symptom. , Cause an increase in the frequency and duration of periods without disease symptoms, or prevent injury or disability due to disease distress. In addition, the terms "effective" and "efficacy" with respect to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy indicates the ability of the drug to promote cancer regression in the patient. Physiological safety indicates the level of toxic or other detrimental physiological effects (side effects) at the cellular, organ and / or organism level upon administration of the drug.

  As an example for the treatment of a tumor, a therapeutically effective amount of the drug is preferably at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, even more preferably as compared to an untreated subject. More preferably, it inhibits cell or tumor growth by at least about 80%. In another preferred embodiment of the invention, tumor regression may be observed and sustained for at least about 20 days, more preferably at least about 40 days, even more preferably at least about 60 days. Despite these final measures of therapeutic efficacy, evaluation of immunotherapeutic drugs must also take into account "immune-related" response patterns.

  The "immune-related" response pattern is a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that produce an anti-tumor effect by inducing a cancer-specific immune response or modifying the natural immune process Is shown. This response pattern is characterized by a beneficial therapeutic effect following the initial increase in tumor burden or the appearance of new lesions, classified as disease progression and synonymous with drug disorders, in the assessment of conventional chemotherapeutic agents . Thus, proper evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on the target disease.

  A therapeutically effective amount of a drug, when administered alone or in combination with an anti-tumor agent, to a subject at risk of developing cancer (eg, a subject having a pre-malignant condition) or a subject having a recurrence of cancer. , A "prophylactically effective amount", which is any amount of a drug that inhibits the development or recurrence of cancer. In a preferred embodiment, the prophylactically effective amount completely prevents the occurrence or recurrence of cancer. "Inhibiting" the occurrence or recurrence of a cancer means either reducing the likelihood of the occurrence or recurrence of the cancer, or completely preventing the occurrence or recurrence of the cancer.

  “Tumor infiltrating inflammatory cells” are any type of cell that generally participates in the inflammatory response in a subject and infiltrates tumor tissue. Such cells include tumor infiltrating lymphocytes (TIL), macrophages, monocytes, eosinophils, histiocytes and dendritic cells.

  The use of an alternative (eg, “or”) should be understood to mean either one, both, or a combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to indicate any described or listed component of "one or more."

  The term "about" or "comprising essentially", as determined by one of ordinary skill in the art, is within the tolerance of a particular value or composition and the method by which the value or composition is measured or determined. Ie, a value or composition that is partially dependent on the limitations of the measurement system. For example, "about" or "comprising essentially" can mean within one or more than one standard deviation throughout implementation in the art. Alternatively, "about" or "comprising essentially" can mean in the range of up to 20%. Furthermore, especially for biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is provided in the present application and claims, the meaning of "about" or "including essentially" is not permitted to the particular value or composition, unless otherwise specified. Should be considered within the error range.

  As described herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise specified, is any integer value within the stated range, and, where appropriate, its fraction ( For example, it should be understood to include one tenth and one hundredth of an integer.

  Various aspects of the invention are described further in the following subsections.

Antibodies of the Invention Abs of the invention include various Abs having the structural and functional properties described herein, including high affinity binding to PD-1 or PD-L1, respectively. These Abs can be used, for example, as therapeutic Abs for treating a subject suffering from a disease, or as reagents in a diagnostic assay for detecting their cognate antigen. Human mAbs (HuMAbs) that specifically bind to PD-1 with high affinity (eg, that can bind to human PD-1 and cross-react with PD-1 from other species, eg, cynomolgus monkeys) HuMAbs that specifically bind to PD-L1 with high affinity are described in US Pat. No. 8,008,449 and US Pat. No. 7,943,743. Abs of the invention include, but are not limited to, all of the anti-PD-1 and anti-PD-L1 Abs described in U.S. Patent Nos. 8,008,449 and 7,943,743, respectively. Other anti-PD-1 mAbs are described, for example, in U.S. Patent Nos. 6,808,710, 7,488,802 and 8,168,757, and PCT Publication No. WO 2012/145493. -PD-L1 mAbs are described, for example, in U.S. Patent Nos. 7,635,757 and 8,217,149, U.S. Publication No. 2009/0317368, and PCT Publication Nos. WO 2011/066389 and WO 2012/145493. ing. To the extent that these anti-PD-1 and anti-PD-L1 mAbs exhibit the structural and functional properties described herein for the antibodies of the present invention, they are also included as antibodies of the present invention.

Anti-PD-1 Antibodies of the Invention Each anti-PD-1 HuMAb described in US Pat. No. 8,008,449 uses one or more of the following characteristics: (a) Biacore biosensor system. to when determined by surface plasmon resonance, binds to human PD-1 with 1x10 ^-7 M or less of _{K D;} (b) does not substantially bind to human CD28, CTLA-4 or ICOS; (c) mixing the lymph Increases T-cell proliferation in a bulb response (MLR) assay; (d) increases interferon-γ production in a MLR assay; (e) increases IL-2 secretion in a MLR assay; (f) human PD-1 And (g) inhibits binding of PD-L1 and / or PD-L2 to PD-1; (h) It has been shown to stimulate antigen-specific memory responses; (i) stimulate Ab responses; and (j) inhibit tumor cell growth in vivo. Anti-PD-1 Abs of the invention include mAbs that specifically bind to human PD-1 and exhibit at least one, and preferably at least five, of the above properties.

U.S. Patent No. 8,008,449 illustrates seven anti-PD-1 HuMAbs: 17D8, 2D3, 4H1, 5C4 (also referred to herein as nivolumab or BMS-936558), 4A11, 7D3 and 5F4. I do. The isolated DNA molecules encoding the heavy and light chain variable regions of these Abs have been sequenced and the amino acid sequences of the variable regions have been deduced. The _VH amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are provided herein as SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7, respectively. The _VL amino acid sequences of 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4 are provided herein as SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14, respectively.

  Preferred anti-PD-1 Abs of the invention include the anti-PD-1 HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. These preferred Abs specifically bind to human PD-1 and (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 1 and SEQ ID NO: A human light chain variable region comprising the contiguously linked amino acids having the sequence set forth in SEQ ID NO: 8; (b) a human heavy chain comprising the contiguously linked amino acids having the sequence set forth in SEQ ID NO: 2 A human light chain variable region comprising a chain variable region and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 9; (c) a continuously linked sequence having the sequence set forth in SEQ ID NO: 3 A human heavy chain variable region comprising a mutated amino acid and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 10; (d) set forth in SEQ ID NO: 4 Array A human heavy chain variable region comprising a contiguously linked amino acid and a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 11; (e) SEQ ID NO: 5 A human heavy chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 12 and a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 12; (F) combining a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 6 and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 13 (G) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 7; Including human light chain variable region comprising the consecutively linked amino acids having a sequence are.

When each of these Abs can bind to PD-1, the _VH and _VL sequences are "mixed and matched" to prepare other anti-PD-1 Abs of the invention. obtain. PD-1 binding of such "mixed and matched" Abs can be determined by binding assays well known in the art, such as enzyme-linked immunosorbent assays (ELISAs), Western blots, radioimmunoassays and Biacore analysis. (See, for example, US Patent No. 8,008,449). Preferably, when the _VH and _VL chains are mixed and matched, the _VH sequence from a particular _VH / _VL pair is replaced with a structurally similar _VH sequence. Similarly, preferably, the _VL sequence from a particular _VH / _VL pair is replaced with a structurally similar _VL sequence. Accordingly, the anti-PD-1 Ab of the present invention comprises (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; An isolated mAb comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13 and 14 or an antigen-binding portion thereof, wherein the Ab is It comprises a mAb or an antigen-binding portion thereof that specifically binds to PD-1, preferably human PD-1.

The CDR domains of the Abs have been expressed using the Kabat system, and these Abs can also be defined by their combination of three heavy and three light chain CDRs (US Pat. No. 8,008, No. 449). Since each of these Abs can bind to PD-1 and antigen binding specificity is primarily provided by the CDR1, CDR2 and CDR3 regions, the _VH CDR1, CDR2 and CDR3 sequences and _Vκ CDR1, CDR2 and The CDR3 sequences can be “mixed and matched” to prepare other anti-PD-1 Abs that also make up the Ab of the invention (ie, CDRs from different Abs can be mixed and matched, of Ab must contain a _V H CDR1, CDR2 and CDR3 and _V κ CDR1, CDR2 and CDR3). The PD-1 binding of such "mixed and matched" Abs can be tested using the binding assays described above, such as ELISA, Western blot, radioimmunoassay and Biacore analysis.

The Abs of the present invention also specifically bind to PD-1 and have a heavy chain variable region from a particular germline heavy chain immunoglobulin and / or a light chain variable region from a particular germline light chain immunoglobulin. And isolated Abs containing Specifically, in one embodiment, the Ab of the present invention comprises: (a) a heavy chain variable comprising contiguously linked amino acids having a sequence derived from a human V _H 3-33 or 4-39 germline sequence; And / or an isolated Ab comprising a light chain variable region comprising contiguously linked amino acids having sequences from human _Vκ L6 or L15 germline sequences. V _{H 3-33,} V H 4-39, amino acid sequences of the _{V H} and V _kappa regions encoded by V _kappa L6 and V _kappa L15 germline gene is provided in U.S. Patent No. 8,008,449 You.

  As used herein, Abs compare the amino acid sequence of a human Ab with the amino acid sequence encoded by a human germline immunoglobulin gene and are closest in sequence to the sequence of the human Ab (ie, the sequence By selecting the best percentage of identity) human germline immunoglobulin sequences, a particular human germline immunoglobulin “derived” can be identified as containing heavy or light chain variable regions. Certain human germ-line immunoglobulin "derived" human Abs have amino acid differences compared to germline sequences, e.g., by the intentional introduction of naturally occurring somatic or site-specific mutations. May be included. However, the selected human Abs will generally be at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and will have germline immunoglobulin amino acid sequences of other species (eg, , A mouse germline sequence) when compared to a human germline sequence. In some cases, a human Ab can be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a germline immunoglobulin gene.

  In one embodiment, the sequence of the human Ab from a particular human germline sequence exhibits up to 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In other embodiments, the human Ab can exhibit a difference of up to 5, or even up to 4, 3, 2, or 1 amino acid from the amino acid sequence encoded by the germline immunoglobulin gene.

Preferred Ab of the present invention is also, (a) a heavy chain variable region comprising the consecutively linked amino acids having a sequence derived from a human V _H 3-33 germline sequence, and from the human V _kappa L6 germline sequence Or (b) a heavy chain variable region comprising contiguously linked amino acids having a sequence derived from a human V _H 4-39 germline sequence. and a light chain variable region comprising consecutively linked amino acids having a sequence derived from a human V _kappa L15 germline sequences, including isolated Ab or antigen binding portion thereof. Examples of V _H 3-33 and V _kappa L6 germline sequence Ab, each having _{V H} and V _kappa derived include 17D8,2D3,4H1,5C4 and 7D3. Examples of Ab having _{V H} and V _kappa regions from V _H 4-39 and V _kappa L15 germline sequences, respectively, including 4A11 and 5F4.

In yet other embodiments, the anti-PD-1 Abs of the present invention are heavy and light chains having amino acid sequences that are highly similar or homologous to the amino acid sequences of preferred anti-PD-1 Abs described herein. A chain variable region, wherein the Ab retains the functional properties of a preferred anti-PD-1 Ab of the present invention. For example, the Ab of the present invention is a mAb comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7. The light chain variable region is selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, 12, 13, and 14 comprising a contiguously linked amino acid having a sequence that is at least 80% identical to the amino acid sequence A mAb comprising consecutively linked amino acids having a sequence that is at least 80% identical to the amino acid sequence to be made. In other embodiments, the _VH and / or _VL amino acid sequence may exhibit at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the above sequence.

  As used herein, the percent sequence identity (also referred to as percent sequence homology) between two sequences (amino acid or nucleotide sequences) is defined by considering the number of gaps and the length of each gap A function of the number of identical positions shared by the sequences relative to the length of the compared sequences, which is introduced to maximize the degree of sequence identity between the two sequences (ie,% identity = identity of identical positions) Number / total number of positions to be compared × 100). Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm well known to those of skill in the art (see, for example, US Pat. No. 8,008,449).

  Antibodies with very similar amino acid sequences may have essentially the same functional properties if the sequence differences are conservative modifications. As used herein, "conservative sequence modification" refers to an amino acid modification that does not significantly affect the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Thus, for example, one or more amino acid residues in the CDR regions of an Ab of the invention can be replaced with other amino acid residues from the same side chain family, and modified Abs are well known in the art. Retained function can be tested using the functional assays provided. Accordingly, one aspect of the anti-PD-1 Abs of the present invention comprises the heavy and light chain variable regions comprising CDR1, CDR2 and CDR3 domains, respectively, wherein one or more of these CDR domains is described herein. Comprising contiguously linked amino acids having the same sequence as the CDR sequence of the preferred anti-PD-1 Abs described (eg, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4) or conservative modifications thereof. The Ab retains the desired functional properties of the preferred anti-PD-1 Ab of the present invention.

  Furthermore, it is well known in the art that heavy chain CDR3 is a major determinant of Ab binding specificity and affinity, and that multiple Abs with the same binding properties based on a common CDR3 sequence can be made as expected. Known (eg, Klimka et al., 2000; Beiboer et al., 2000; Rader et al., 1998; Barbas et al., 1994; Barbas et al., 1995; Ditzel et al., 1996; Berezov et al., 2001; Igarashi et al., 1995; Bourgeois et al., 1998; Levi et al., 1993; Polymenis and Stoller, 1994; and Xu and Davis, 2000). The literature generally demonstrates that when the heavy chain CDR3 sequence of a given Ab is defined, variability in the other 5 CDR sequences does not significantly affect the binding specificity of the Ab. Thus, an Ab of the invention comprising six CDRs can be defined by specifying the sequence of the heavy chain CDR3 domain.

  The anti-PD-1 Ab of the present invention also specifically binds to human PD-1 and binds to human PD-1 with any of HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 and 5F4. And isolated Abs that cross-compete. Thus, the anti-PD-1 Abs of the present invention are characterized in that, for binding to PD-1, (a) a human heavy chain comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 1 A human light chain variable region comprising a variable region and a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 8; (b) a continuously linked sequence having the sequence set forth in SEQ ID NO: 2 A human heavy chain variable region comprising a continuous amino acid having the sequence set forth in SEQ ID NO: 9 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 9; A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 10; and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 10; Described in 4 A human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 11; (e) A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 5 and a human light chain comprising the continuously linked amino acid having the sequence set forth in SEQ ID NO: 12 A chain variable region; (f) a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 6 and a continuously linked sequence having the sequence set forth in SEQ ID NO: 13. (G) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 7 and SEQ ID NO: 1 4. An isolated Ab or an antigen binding portion thereof that cross-competes with a reference Ab or a reference antigen binding portion thereof, comprising a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in No. 4. .

  The ability of Abs to cross-compete for binding to an antigen is that these Abs bind to the same epitope region of the antigen (ie, the same or overlapping or adjacent epitopes) and other cross-competing Abs to a particular epitope region Sterically hinders the binding of Thus, the ability of a test Ab to competitively inhibit the binding of, for example, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3, or 5F4 to human PD-1, indicates that the test Ab is 17D8, 2D3, 4H1, 5C4, 4A11. , 7D3 or 5F4, respectively, and the same epitope region of human PD-1. All isolated Abs that bind HuMAbs 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 and the same epitope region of human PD-1 are included in the Abs of the invention. These cross-competing Abs are expected to have very similar functional properties due to the binding of PD-1 to the same epitope region. For example, the cross-competing anti-PD-1 mAbs 5C4, 2D3, 7D3, 4H1 and 17D8 have been shown to have similar functional properties (US Pat. No. 8,008,449, Examples 3-7). reference). The higher the degree of cross-competition, the more similar functional characteristics. In addition, Abs that cross-compete can be easily identified based on their ability to cross-compete with 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 in a standard PD-1 binding assay. For example, Biacore analysis, ELISA assays or flow cytometry can be used to demonstrate cross-competition with Abs of the invention (see, eg, Examples 1 and 2).

  In one embodiment, 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 cross-competes for binding to human PD-1, or 17D8, 2D3, 4H1, 5C4, 4A11, 7D3 or 5F4 and human Abs that bind to the same epitope region of PD-1 are mAbs. For administration to human patients, these cross-competing Abs are preferably chimeric Abs, or more preferably humanized or human Abs. Such human mAbs can be prepared and isolated as described in US Patent No. 8,008,449. The data provided in Example 1 shows that 5C4 or its Fab fragment cross-competes with 2D3, 7D3, 4H1 or 17D8, respectively, for binding to hPD-1 expressed on the surface of cells. Shows that all five anti-PD-1 mAbs bind to the same epitope region of hPD-1 (FIGS. 1A-1C).

The anti-PD-1 Abs of the present invention may further comprise one or more of the V (s) described herein as a starting material, such as to manipulate a modified Ab that may have altered properties from the starting Ab. It can be prepared using Abs having _H and / or _VL sequences. An Ab may include one or more of one or more variable regions (ie, V _H and / or V _L ), for example, one or more CDR regions and / or one or more framework regions. It can be manipulated by modifying the residues. Additionally or alternatively, Abs can be engineered, for example, by modifying residues in the constant region to alter the effector function of the Ab. Certain modifications to Ab is, CDR grafting, one or more binding properties of Ab (e.g., affinity) of the _{V H} and / or V _kappa CDRl, CDR2 and / or amino acid residues in the CDR3 regions to improve the Site-directed mutation, a site-directed mutation of an amino acid residue within the _VH and / or _Vκ framework regions so as to reduce the immunogenicity of the Ab, generally one or more functional properties of the Ab, For example, modifications in the Fc region to alter serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cytotoxicity, and increase the biological (eg, serum) half-life of the Ab Or a chemical modification to reduce it, eg, an alteration in the pegylation or glycosylation pattern. Specific examples of such modifications and methods of manipulating Abs are described in detail in US Pat. No. 8,008,449. The anti-PD-1 Abs of the present invention include all such engineered Abs that specifically bind to human PD-1 and are obtained by modification of any of the above anti-PD-1 Abs.

The anti-PD-1 Ab of the present invention also includes the antigen-binding portion of the Ab. It has been demonstrated in detail that the antigen-binding function of the Ab can be performed by fragments of the full-length Ab. Examples of binding fragments included within the term “antigen binding portion” of an Ab are: (i) a Fab fragment that is a monovalent fragment consisting of the V _L , V _H , C _L and C _H1 domains; F (ab ') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at: (iii) a Fd fragment consisting of the _VH and _CH1 domains; and (iv) a single Ab Includes an Fv fragment consisting of the V _L and V _H domains of the arm.

These fragments, first obtained via proteolysis with enzymes such as papain and pepsin, are then engineered into monovalent and multivalent antigen binding fragments. For example, monovalent although _{V L} and _{V H} of the two domains of the Fv fragment are encoded by separate genes, they, _{V L} and _{V H} regions is paired, known as single chain variable fragment (scFv) Can be linked using recombinant methods, with a synthetic linker peptide that allows it to be made as a single protein chain to form a molecule of. Divalent or bivalent scFvs (di-scFv or bi-scFv) are two scFvs in a single peptide chain known as a tandem scFv containing two _VH and two _VL regions. Can be operated by connecting. Use a linker peptide of less than 10 amino acids that is too short to fold together for the two variable regions so as to dimerize the scFv and produce diabodies or form other multimers. Thus, ScFv dimers and higher multimers can also be produced. Diabodies have a maximum 40-fold lower dissociation constant than the K _D values for scFv, it has been shown to bind to the cognate antigen with much higher affinity than the corresponding scFv. Very short linkers (≦ 3 amino acids) resulted in the formation of a trivalent triabody or tetravalent tetrabody that shows even higher affinity for the antigen than a diabody. Other variants include _{scFv-C H3} dimer in which minibodies, and the larger scFv-Fc fragment _(scFv-C H2 _{-C H3} dimer), Moreover, an isolated CDR may exhibit antigen-binding function . These Ab fragments are engineered using conventional recombinant techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as the intact Ab. All of the above proteolytic and engineered fragments of Ab and related variants (see Hollinger and Hudson, 2005; Olafsen and Wu, 2010 for further details) are within the term "antigen binding portion" of Ab. It is intended to be included.

Anti-PD-L1 Antibodies of the Invention Each of the anti-PD-L1 HuMAbs described in U.S. Pat. No. 7,943,743 has one or more of the following properties (a) a K of 1 × 10 ⁻⁷ M or less. _Binds human PD-L1 at _D ; (b) increases T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increases interferon-γ production in an MLR assay; (d) in an MLR assay Increases IL-2 secretion; (e) stimulates Ab response; (f) inhibits binding of PD-L1 to PD-1; and (g) T cells against T cell effector cells and / or dendritic cells Has been shown to reverse the inhibitory effect of regulatory cells. Anti-PD-L1 Abs of the invention include mAbs that specifically bind to human PD-L1 and exhibit at least one, and preferably at least four, of the above properties.

U.S. Pat. No. 7,943,743 discloses ten anti-PD-1 HuMAbs: 3G10, 12A4 (also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4 are illustrated. The isolated DNA molecules encoding the heavy and light chain variable regions of these Abs have been sequenced and the amino acid sequences of the variable regions have been deduced. 3G10,12A4,10A5,5F8,10H10,1B12,7H1,11E6,12B7 and each _{V H} amino acid sequences of 13G4, shown in SEQ ID NO: 15,16,17,18,19,20,21,22,23 and 24 And their _VL amino acid sequences are shown in SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, respectively.

  Preferred anti-PD-L1 Abs of the present invention include the anti-PD-L1 HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4. These preferred Abs specifically bind to human PD-L1 and (a) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 15 and SEQ ID NO: A human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 25; (b) a human heavy chain comprising continuously linked amino acids having the sequence set forth in SEQ ID NO: 16. A human light chain variable region comprising a chain variable region and a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 26; (c) a continuously linked sequence having the sequence set forth in SEQ ID NO: 17 A human heavy chain variable region comprising a mutated amino acid and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 27; A human heavy chain variable region comprising a contiguously linked amino acid having the sequence set forth and a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 28; (e) A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 19 and a human light chain comprising the continuously linked amino acids having the sequence set forth in SEQ ID NO: 29 A chain variable region; (f) a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 20 and a continuously linked sequence having the sequence set forth in SEQ ID NO: 30. (G) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 21 and SEQ ID NO: A human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 1; (h) a human heavy chain comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 22. A human light chain variable region comprising a chain variable region and a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 32; (i) a continuously linked sequence having the sequence set forth in SEQ ID NO: 23 A human heavy chain variable region comprising a mutated amino acid and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 33; or (j) And a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 34. including.

When each of these Abs can bind to PD-L1, the _VH and _VL sequences can be "mixed and adapted" to prepare other anti-PD-L1 Abs of the invention. The PD-L1 binding of such “mixed and matched” Abs can be determined by binding assays well known in the art, such as ELISA, Western blot, radioimmunoassay and Biacore analysis (see, eg, US Pat. No. 7,943,743). Preferably, when the _VH and _VL chains are mixed and matched, the _VH sequence from a particular _VH / _VL pair is replaced with a structurally similar _VH sequence. Similarly, preferably, the _VL sequence from a particular _VH / _VL pair is replaced with a structurally similar _VL sequence. Accordingly, the Abs of the present invention also include contiguously linked amino acids having the sequence set forth in any of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. A light chain variable comprising a heavy chain variable region and a continuously linked amino acid having a sequence set forth in any of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 A region-containing mAb or antigen-binding portion thereof, wherein the Ab comprises a mAb that specifically binds to PD-L1, preferably human PD-L1, or an antigen-binding portion thereof.

The CDR domains of the anti-PD-L1 HuMAbs have been expressed using the Kabat system, and these Abs can also be defined by a combination of their three heavy and three light chain CDRs (US Pat. No. 7,943,743). Snce Each of these Abs is capable of binding to PD-L1 and the antigen binding specificity is mainly provided by the CDR1, CDR2 and CDR3 regions, so that the _VH CDR1, CDR2 and CDR3 sequences and _Vκ CDR1, CDR2 And CDR3 sequences can be "mixed and matched" to prepare other anti-PD-1 Abs that also make up the Abs of the invention (i.e., CDRs from different Abs can be mixed and matched, each Ab must contain a _V H CDR1, CDR2 and CDR3 and _V κ CDR1, CDR2 and CDR3). The PD-L1 binding of such “mixed and matched” Abs can be tested using, for example, ELISAs, Western blots, radioimmunoassays and Biacore analysis.

The antibodies of the present invention also specifically bind to PD-L1 and have a heavy chain variable region derived from a particular germline heavy chain immunoglobulin and / or a light chain variable region derived from a particular germline light chain immunoglobulin. And isolated Abs containing Specifically, in one aspect, the Abs of the invention are (a) sequentially linked having sequences from human _VH 1-18, 1-69, 1-3 or 3-9 germline sequences. including Ab comprising a light chain variable region comprising the heavy chain variable region, and / or human V _kappa L6, L15, consecutively linked amino acids having a sequence derived from A27 or L18 germline sequence comprises the amino acid . _{V H 1-18, V H 1-3,} V H 1-69, V H 3-9, V κ L6, V κ L15 and V _kappa A27 _{V H} and V _kappa regions encoded by germline gene The amino acid sequence is provided in U.S. Patent No. 7,943,743.

Preferred Ab of the present invention, (a) a heavy chain variable region comprising the consecutively linked amino acids having a sequence derived from a human V _H 1-18 germline sequence, and from a human V _kappa L6 germline sequence A light chain variable region comprising a continuously linked amino acid having a sequence; (b) a heavy chain variable region comprising a continuously linked amino acid having a sequence derived from a human V _H 1-69 germline sequence; and It is continuously connected with a sequence from (c) human V _H 1-3 germline sequence; light chain variable region comprising the consecutively linked amino acids having a sequence derived from a human V _kappa L6 germline sequence heavy chain variable region and a light chain variable region comprising the consecutively linked amino acids having a sequence derived from a human V _kappa L15 germline sequence comprises the amino acid; (d) human V _H 1-69 germline A light chain variable region comprising the continuous heavy chain variable region comprising the linked amino acids and consecutively linked amino acids having a sequence derived from a human V _kappa A27 germline sequence, having the sequence from columns; (e ) is continuously connected with a sequence derived from a human V _H 3-9 heavy chain variable region comprising the consecutively linked amino acids having a sequence derived from germline sequence, and the human V _kappa L15 germline sequence or (f) a heavy chain variable region, and a human V _kappa L18 germline sequence comprises continuously linked amino acids having a sequence derived from a human V _H 3-9 germline sequence; a light chain variable region comprising the amino acid An isolated Ab or antigen-binding portion thereof comprising a light chain variable region comprising contiguously linked amino acids having a sequence of origin.

Examples of V _H 1-18 and V _kappa L6 germline sequence Ab, each having _{V H} and V _kappa derived is 3G10. Examples of Ab having _{V H} and V _kappa regions from V _H 1-69 and V _kappa L6 germline sequences, respectively, including 12A4,1B12,7H1 and 12B7. Examples of V _H 1-3 and V _kappa L15 germline sequence Ab, each having _{V H} and V _kappa derived is 10A5. Examples of V _H 1-69 and V _kappa A27 Ab having _{V H} and V _kappa regions from each germline sequence comprises 5F8,11E6 and 11E6a. Examples of V _H 3-9 and V _kappa L15 germline sequence Ab, each having _{V H} and V _kappa derived is 10H10. Examples of V _H 1-3 and V _kappa L15 germline sequence Ab, each having _{V H} and V _kappa derived is 10A5. Examples of V _H 3-9 and V _kappa L18 germline sequence Ab, each having _{V H} and V _kappa derived is 13G4.

In one embodiment, the anti-PD-L1 Ab of the present invention comprises a heavy and light chain having an amino acid sequence that is highly similar or homologous to the amino acid sequence of a preferred anti-PD-L1 Ab described herein. A chain variable region, wherein said Ab retains the functional properties of said anti-PD-L1 Ab of the invention. For example, the Ab of the present invention is a mAb containing a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 comprising a contiguously linked amino acid having a sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, And mAbs comprising consecutively linked amino acids having a sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of 31, 32, 33 and 34. In other embodiments, the _VH and / or _VL amino acid sequence may exhibit at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the above sequence.

  One aspect of the anti-PD-L1 Ab of the present invention comprises heavy and light chain variable regions comprising CDR1, CDR2 and CDR3 domains, respectively, wherein one or more of these CDR domains is described herein. Are continuously linked having the same sequence as the CDR sequence of a preferred anti-PD-L1 Ab (eg, 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4) or a conservative modification thereof. The Ab retains the desired functional properties of the preferred anti-PD-L1 Ab of the present invention.

  Based on the evidence that heavy chain CDR3 is a major determinant of Ab binding specificity and affinity, generally, when the heavy chain CDR3 sequence for a given Ab is defined, the variable in the other five CDR sequences Does not significantly affect the binding specificity of the Ab. Thus, the anti-PD-L1 Abs of the present invention include six CDRs and include isolated Abs defined by specifying the sequence of the heavy chain CDR3 domain.

  The anti-PD-L1 Ab of the present invention also specifically binds to human PD-L1 and has a HuMAb 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6 binding to human PD-L1. , 12B7 and 13G4. Accordingly, the anti-PD-L1 Abs of the present invention are characterized in that, for binding to PD-L1, (a) a human heavy chain comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 15 A human light chain variable region comprising a variable region and a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 25; (b) a continuously linked sequence having the sequence set forth in SEQ ID NO: 16 A human heavy chain variable region comprising the amino acid sequence and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 26; (c) the sequence set forth in SEQ ID NO: 17 A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 27 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 27; : A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 18 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 28 A region; (e) a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 19 and a continuously linked region having the sequence set forth in SEQ ID NO: 29. A human light chain variable region comprising amino acids; (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 20 and the sequence set forth in SEQ ID NO: 30. A human light chain variable region comprising contiguously linked amino acids having a sequence set forth in SEQ ID NO: 21; And a light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 31; (h) a continuously linked amino acid having the sequence set forth in SEQ ID NO: 22 A human heavy chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 32 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 32; A human heavy chain variable region comprising contiguously linked amino acids and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 33; or (j) SEQ ID NO: 24 A human heavy chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 34 and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 34 DOO comprises a light chain variable region, including the reference Ab or isolated Ab or antigen binding portion thereof cross-competes with a reference antigen-binding portion thereof.

  The ability of an Ab to cross-compete with any of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 for binding to human PD-L1 indicates that such Abs are 3G10, 12A4, 10A5, 5F8. , 10H10, 1B12, 7H1, 11E6, 12B7 and 13G4, respectively. All isolated Abs that bind to the same epitope region of HuMAbs 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4 and human PD-L1 are included in the Abs of the present invention. These cross-competing Abs are expected to have very similar functional properties due to the binding of PD-L1 to the same epitope region. For example, the cross-competing anti-PD-L1 mAbs 3G10, 1B12, 13G4, 12A4 (BMS-936559), 10A5, 12B7, 11E6 and 5F8 have been shown to have similar functional properties (US Pat. 7,943,743, Example 3-11), mAb 10H10 binding to different epitope regions functions differently (US Patent No. 7,943,743, Example 11). The higher the degree of cross-competition, the more similar functional characteristics. Further, cross-competing Abs can be identified in standard PD-L1 binding assays well known to those of skill in the art, such as Biacore analysis, ELISA assays or flow cytometry. In a preferred embodiment, 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7 or 13G4 cross-competes for binding to human PD-1, or 3G10, 12A4, 10A5, 5F8, 10H10, The Ab that binds to the same epitope region of 1B12, 7H1, 11E6, 12B7 or 13G4 and human PD-L1 is a mAb, preferably a chimeric Ab, or more preferably a humanized or human Ab. Such human mAbs can be prepared and isolated as described in U.S. Patent No. 7,943,743.

  The data provided in Example 2 indicates that each of the anti-PD-L1 HuMAbs 5F8, 7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7, and 13G4, ie, all of the tested HuMAbs except 10H10 had PD -Indicates that binding of mAbs 3G10, 10A5, 11E6, 12A4 and 13G4 to Chinese hamster ovary (CHO) cells expressing L1 cells was substantially blocked. HuMAb 10H10 substantially blocked only its own binding to CHO / PD-L1 cells. These data show that 3G10, 10A5, 11E6, 12A4, and 13G4 cross-compete with all of the tested HuMAbs except 10H10 for binding to the same epitope region of human PD-L1 (FIG. 2A). -F).

  The data provided in Example 3 shows that the binding of HuMAb 12A4 to ES-2 ovarian carcinoma cells expressing PD-L1 cells was substantially blocked by 12A4 itself or by 1B12 and 12B7, and mAb 5F8, Indicates that it was moderately to significantly blocked by 10A5, 13G4 and 3G10, but not mAb 10H10. These data, which are in great agreement with the data in Example 2, indicate that 12A4 itself, as well as two other HuMabs, 12B7 and 1B12, bind to the same epitope region of human PD-L1, possibly to the same epitope. 5F8, 10A5, 13G4 and 3G10 show cross-competition with 12A4 at lower but significant levels, and that these mAbs can bind to epitopes that overlap with the 12A4 epitope. However, it shows that 10H10 did not cross-compete with 12A4 at all (FIG. 3), suggesting that this mAb binds to a different epitope region than 12A4.

An anti-PD-L1 Ab of the present invention is also an Ab engineered starting from an Ab having one or more _VH and / or _VL sequences described herein, wherein Includes engineered Abs that may have altered properties. The anti-PD-L1 Ab can be manipulated with the various modifications described above for the manipulation of the modified anti-PD-1 Ab of the present invention.

  Anti-PD-L1 Abs of the invention also include isolated Abs selected for their ability to bind to PD-L1 in formalin-fixed paraffin-embedded (FFPE) tissue specimens. The use of FFPE samples is important for long-term follow-up analysis of the correlation between PD-L1 expression in tumors and disease prognosis or progression. In addition, tests in measuring PD-L1 expression generally include anti-human PD-L1 Ab (Hamanishi et al., 2007), which can be used to stain PD-L1 in FFPE specimens by IHC; In particular, it is often performed in frozen specimens due to difficulties in isolating Abs that specifically bind to the membrane PD-L1 in these tissues. The use of different Abs to stain PD-L1 in frozen versus FFPE tissues, and the ability of certain Abs to distinguish between the membrane and / or cytoplasmic forms of PD-L1 indicates that PD-L1 expression is associated with disease prognosis Could explain some of the different data reported in the literature (Hamanishi et al., 2007; Gadiot et al., 2011). The present application provides several rabbit mAbs that specifically bind with high affinity to human PD-L1 in membranes in FFPE tissue samples containing tumor cells and tumor infiltrating inflammatory cells.

  Rabbit and mouse anti-hPD-L1 mAbs were produced as described in Example 9. Of the approximately 200 Ab multiclones screened, only 10 rabbit multiclones Ab were found to specifically detect the membrane morphology of PD-L1 and the top 5 multiclones (designated number 13, 20, 28, 29 and 49) were then subcloned. The clone that produced the most specific detection of PD-L1 in the membrane, rabbit clone 28-8, was selected for the IHC assay. The sequence of the variable region of mAb 28-8 is set forth in SEQ ID NOs: 35 and 36, respectively. When represented using the Kabat system, the sequences of the heavy and light chain CDR domains of mAb 28-8 are set forth in SEQ ID NOs: 37-42. Rabbit clones 28-1, 28-12, 29-8 and 20-12 were the next best mAbs for strong detection of PD-L1 on membranes in FFPE tissues.

The anti-PD-L1 Ab of the present invention may also comprise Fab, F (ab ') ₂ , Fd, Fv, and scFv, di-scFv or bi-scFv, and scFv-Fc fragments, diabodies, triabodies, tetrabodies. And the antigen-binding portion of the Ab, including the isolated CDRs (see Hollinger and Hudson, 2005; Olafsen and Wu, 2010 for more details).

Nucleic acid molecules encoding the antibodies of the invention Another aspect of the present specification relates to isolated nucleic acid molecules encoding any of the Abs of the invention. These nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. The nucleic acids of the invention can be, for example, DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA.

  The nucleic acids of the invention can be obtained using standard molecular biology techniques. In Abs expressed by hybridomas (eg, hybridomas prepared from transgenic mice having the human immunoglobulin genes described further below), the cDNAs encoding the light and heavy chains of the Abs produced by the hybridomas are: , Standard PCR amplification or cDNA cloning techniques. Ab-encoding nucleic acids obtained from an immunoglobulin gene library (eg, using phage display technology) can be recovered from the library.

Preferred nucleic acid molecules of the present invention, anti -PD-1 HuMAb, those encoding the _{V H} and V _kappa sequences 17D8,2D3,4H1,5C4,4A11,7D3 and 5F4 (U.S. Patent No. 8,008,449 are described), and anti -PD-L1 HuMAb, those encoding the _{V H} and V _kappa sequences 3G10,12A4,10A5,5F8,10H10,1B12,7H1,11E6,12B7 and 13G4 (U.S. Patent No. 7, 943, 743). The isolated DNA encoding the V _H region, _V DNA encoding _H, are known in the art, the heavy chain constant region is a sequence that can be obtained by standard PCR amplification _{(C H1, C H2} And operably linked to another DNA molecule encoding C _H3 ), which can be converted to a full-length heavy chain gene. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgG1 or IgG4 constant region. Similarly, V isolated DNA encoding the _L region, the DNA encoding the V _L, are known in the art, the light chain constant region (C _L which is a sequence that can be obtained by standard PCR amplification ) Can be converted to a full-length light chain gene by operably linking it to another DNA molecule encoding The light chain constant region can be a kappa or lambda constant region, but is more preferably a kappa constant region.

Pharmaceutical Compositions The antibodies of the present invention may constitute a composition, eg, a pharmaceutical composition, comprising one Ab or a combination of Abs, or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and physiologically compatible. Including absorption delaying agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). The pharmaceutical compositions of the invention may include one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. .

  Dosage regimens are adjusted to provide the optimum desired response, eg, a therapeutic response or minimal side effects. For administration of an anti-PD-1 or anti-PD-L1 Ab, the dose may range from about 0.0001 to about 100 mg / kg of the subject's body weight, usually from about 0.001 to about 20 mg / kg of the subject's body weight, more usually about 0 to about 100 mg / kg of the subject. Ranging from .01 to about 10 mg / kg of the subject's body weight. Preferably, the dose will be in the range of 0.1-10 mg / kg body weight. For example, the dose may be 0.1, 0.3, 1, 3, 5, or 10 mg / kg body weight, more preferably 0.3, 1, 3, 3 or 10 mg / kg body weight. Dosage schedules are generally designed to achieve exposure that causes sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the Ab. A typical treatment regimen is once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every March, or once every March to June. Requires multiple administrations. Dosage and scheduling may be changed during the course of the treatment. For example, the dosing schedule is: (i) every 2 weeks in a 6 week cycle; (ii) 6 doses every 4 weeks, then every 3 months; (iii) every 3 weeks; (iv) once every 3- This may include administering the Ab at 10 mg / kg body weight, then at 1 mg / kg body weight every 2-3 weeks. Given that IgG4 Abs generally have a half-life of 2-3 weeks, a preferred dosing regimen for an anti-PD-1 or anti-PD-L1 Ab of the present invention will provide a complete response or an established progressive 0.3 to 10 mg / kg body weight via intravenous administration, preferably 3 to 10 mg / kg body weight, more preferably 3 mg / kg body weight, for Abs given every 14 days in up to 6 or 12 week cycles until disease Including.

  In some methods, two or more mAbs with different binding specificities are administered simultaneously, provided that the dose of each Ab administered is within a specified range. Antibodies are usually administered on multiple occasions. The interval between single doses can be, for example, every week, every two weeks, every three weeks, every month, every three months, or every year. Intervals can also be irregular as indicating the need by measuring blood levels of Ab against target antigen in the patient. In some methods, the dose is adjusted to achieve a plasma Ab concentration of about 1-1000 μg / ml, and in some methods, about 25-300 μg / ml.

  Alternatively, Abs can be administered as a sustained release formulation in cases where less frequent administration is required. Dosage and frequency will vary depending on the half-life of the Ab in the patient. Generally, human Abs have the longest half-life, followed by humanized Abs, chimeric Abs, and non-human Abs. The dose and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are generally administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses are sometimes required at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient shows a partial or complete improvement in the symptoms of the disease. . Thereafter, the patient can be administered a prophylactic regime.

  The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention will be effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without undue toxicity to the patient. It can be varied to obtain the amount of the active ingredient. The selected dosage level will depend on various pharmacokinetic factors, such as the activity of the particular composition of the invention used, the route of administration, the time of administration, the elimination rate of the particular compound used, the duration of the treatment, Other drugs, compounds and / or substances used in combination with certain compositions, the age, sex, weight, condition, general health and past medical history of the treated patient, and factors well known in the pharmaceutical arts. And so on. The compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of well-known methods in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result.

Uses and Methods of the Invention The Abs, Ab compositions, nucleic acids and methods of the invention have numerous in vitro and in vivo utilities, such as binding Abs to target polypeptides, or encode these polypeptides. A method for determining and quantifying the expression of PD-1 or PD-L1, comprising measuring the amount of a nucleic acid, and a composition comprising a therapeutically effective amount of a therapeutic agent that inhibits signaling from an inhibitory immune regulator A method for immunotherapy of a subject suffering from a disease, comprising administering to a subject. In a preferred embodiment of the latter method, the inhibitory immunoregulator is a component of the PD-1 / PD-L1 signaling pathway, and the therapeutic agent disrupts signaling of this pathway. More preferably, the therapeutic agent is an Ab that blocks the interaction between PD-1 and PD-L1. In one preferred embodiment of this method, the Ab specifically binds to PD-1 and blocks the interaction of PD-1 with PD-L1 and / or PD-L2. In another preferred embodiment, the therapeutic agent is an Ab that specifically binds to PD-L1 and blocks the interaction of PD-L1 with PD-1 and / or B7-1 (CD80). Accordingly, the present application enhances the immune response in a subject, including administering an anti-PD-1 and / or anti-PD-L1 Ab to disrupt the interaction between PD-1 and PD-L1. And methods for treating diseases mediated by such enhancement of the immune response. When the Abs for PD-1 and PD-L1 are administered together, the two can be administered sequentially or simultaneously in any order. In one aspect, the application relates to administering to a subject an anti-PD-1 and / or anti-PD-L1 Ab of the invention, or an antigen-binding portion thereof, such that the immune response in the subject is modified. A method of modifying an immune response in a subject is provided. Preferably, the immune response is enhanced, augmented, stimulated, or up-regulated. In a preferred embodiment, the Ab of the invention is a human Ab.

  Preferred subjects include human patients in need of an increased immune response. The immunotherapeutic methods described herein are particularly suitable for treating a human patient having a disorder that can be treated by enhancing a T cell-mediated immune response. In one embodiment, the method is used for treating a subject suffering from a disease caused by an infectious agent. In a preferred embodiment, the method is used for treating a subject that has, or is at risk of having, cancer.

Cancer immunotherapy Blocking the PD-1 / PD-L1 interaction enhances the immune response in vitro (U.S. Pat. Nos. 8,008,449 and 7,943,743; Fife et al., 2009) and preclinical antitumor It has been shown to mediate activity (Dong et al., 2002; Iwai et al., 2002). However, the molecular interactions that can be blocked by these two Abs are not identical: the anti-PD-1 Abs of the present invention destroy PD-1 / PD-L1 and PD-1 / PD. May disrupt the L2 interaction; in contrast, the anti-PD-L1 Abs of the invention also disrupt the PD-1 / PD-L1 interaction, whereas they do Does not block the / PD-L2 interaction, but may instead disrupt the PD-1-independent PD-L1 / CD80 interaction and has been shown to down-regulate T-cell responses in vitro and in vivo (Park et al., 2010; Paterson et al., 2011; Yang et al., 2011; Butte et al., 2007; Butte et al., 2008). Thus, among these various ligand-receptor pairs, different interactions may dominate in different cancer types and contribute to different activity profiles for the two Abs.

  Disruption of the PD-1 / PD-L1 interaction by an antagonistic Ab can increase the immune response to cancerous cells in a patient. PD-L1 is not expressed in normal human cells, but is abundant in various human cancers (Dong et al., 2002). The interaction between PD-1 and PD-L1 attenuates the T cell response as manifested in a decrease in tumor infiltrating lymphocytes (TIL) and an increase in T cell receptor mediated proliferation, T cell anergy, depletion or apoptosis, and Causes immune evasion by cancerous cells (Zou and Chen, 2008; Blank et al., 2005; Konishi et al., 2004; Dong et al., 2003; Iwai et al., 2002). Immunosuppression can be reversed by using anti-PD-1 and / or anti-PD-L1 Ab to inhibit local interactions between PD-L1 and PD-1. These Abs can be used alone or in combination to inhibit the growth of cancerous tumors. In addition, either or both of these Abs can be used with other immunogenic and / or anti-cancer agents such as cytokines, standard cancer chemotherapy, vaccines, radiation therapy, surgery or other Abs.

The present application relates to a method for immunotherapy of a subject suffering from cancer, comprising PD-1 and PD-L1 and / or PD-L2. And administering to the subject a therapeutically effective amount of the Ab or an antigen-binding portion thereof that disrupts the interaction of The application also includes administering to a subject an Ab or an antigen-binding portion thereof that disrupts the interaction of PD-1 with PD-L1 and / or PD-L2 in an amount effective to inhibit tumor cell growth. A method for inhibiting the growth of tumor cells in a subject is provided. In a preferred embodiment, the subject is a human. In another preferred embodiment, the Ab or antigen binding portion thereof is an anti-PD-1 Ab of the invention or antigen binding portion thereof. In one embodiment, the Ab or antigen binding portion thereof is of the IgG1 or IgG4 isotype. In other embodiments, the Ab or antigen binding portion thereof is a mAb or antigen binding portion thereof. In a further aspect, the Ab or antigen binding portion thereof is a chimeric, humanized or human Ab or antigen binding portion thereof. In a preferred embodiment for treating a human patient, the Ab or antigen binding portion thereof is a human Ab or antigen binding portion thereof.

The clinical trial described in the examples used the anti-PD-1 HuMA, nivolumab (designated 5C4 in U.S. Patent No. 8,008,449) to treat cancer. Although 5C4 was selected as the lead Ab for participation in clinical trials, it is worth noting that some of the anti-PD-1 Abs of the present invention have 5C4 functional properties that are important for 5C4's therapeutic activity, such as, for example, Specific binding with high affinity to human PD-1, increasing T-cell proliferation, IL-2 secretion and interferon-γ production in MLR assays, PD-L1 and / or PD to PD-1 -Sharing L2 binding and inhibiting tumor cell growth in vivo. Furthermore, certain anti -PD-1 Ab of the present invention, 17D8,2D3,4H1 and 7D3 are the _{V H} and V _kappa region having a sequence derived from the _V H 3-33 and V _kappa L6 germline sequence, respectively And structurally related to 5C4. In addition, 5C4, 2D3, 7D3, 4H1 and 17D8 all cross-compete for binding to the same epitope region of hPD-1 (Example 1). Thus, the preclinical characterization of nivolumab and other anti-PD-1 HuMabs indicates that the methods for treating cancer provided herein are selected from a wide variety of anti-PD-1 Abs of the present invention. Of the present invention.

Accordingly, one aspect of the immunotherapy methods described herein comprises: (a) a heavy chain variable region comprising contiguously linked amino acids having a sequence derived from a human V _H 3-33 germline sequence , and continuous with the sequences from the light chain variable region, or (b) human V _H 4-39 germline sequence comprises continuously linked amino acids having a sequence derived from a human V _kappa L6 germline sequence heavy chain variable region and anti -PD-1 Ab or antigen comprises a light chain variable region comprising the consecutively linked amino acids having a sequence derived from a human V _kappa L15 germline sequences, including linked amino acids Administering the binding moiety to the patient.

  In one other embodiment, the Ab or antigen-binding portion thereof administered to the patient is continuously linked for binding to PD-1 (a) having the sequence set forth in SEQ ID NO: 1. A human heavy chain variable region comprising the amino acid sequence and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 8; (b) the sequence set forth in SEQ ID NO: 2 A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 9 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 9; 3. A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 3 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 10. (D) a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 4 and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 11 (E) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 5 and a contiguous sequence having the sequence set forth in SEQ ID NO: 12. A human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 6; and a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 6 and SEQ ID NO: 13. A human light chain variable region comprising a contiguously linked amino acid having the sequence set forth; or (g) a human comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 7. Chain variable region and SEQ ID NO: 14 comprises a human light chain variable region comprising the consecutively linked amino acids having the sequence set forth in a cross-compete reference Ab or reference antigen binding portion thereof. In a preferred embodiment, the Ab or antigen binding portion thereof cross-competes with nivolumab for binding to PD-1.

  In one embodiment of the immunotherapy methods described herein, the anti-PD-1 Ab or antigen-binding portion thereof administered to the patient has (a) the sequence set forth in SEQ ID NO: 1. A human heavy chain variable region comprising contiguously linked amino acids and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 8; (b) SEQ ID NO: 2 A human heavy chain variable region comprising a contiguously linked amino acid having the described sequence and a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 9; c) a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 3 and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 10 (D) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 4 and a contiguous sequence having the sequence set forth in SEQ ID NO: 11. (E) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 5 and a human light chain variable region comprising the sequence set forth in SEQ ID NO: 5; A human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 6; and (f) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 6. And a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 13; or (g) continuously linked having the sequence set forth in SEQ ID NO: 7 And a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 14. In a preferred embodiment, the anti-PD-1 Ab or antigen binding portion is a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 4 and set forth in SEQ ID NO: 11. A human light chain variable region comprising contiguously linked amino acids having the sequence In another preferred embodiment, the anti-PD-1 Ab is nivolumab.

  In one embodiment of the present method, the anti-PD-1 Ab is formulated for intravenous administration. In a preferred embodiment, the Ab is administered intravenously at a dose of 3 mg / kg every two weeks for 60 minutes. Generally, treatment is continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

  In the clinical trials of anti-PD-1 immunotherapy described in the Examples below, interesting ORs with a sustained clinical response were significantly greater in NSCLC, MEL and RCC patients, even in well-pretreated patients. Across a large proportion of multiple tumor types, they were observed at various sites of metastasis, including liver, lung, lymph nodes and bone. MEL and RCC are used in cancer immunotherapy, such as interferon-alpha and interleukin-2 (Eton et al., 2002; Coppin et al., 2005; McDermott and Atkins, 2006) in both MEL and RCC and anti-CTLA-4 in MEL. It is believed to be an immunogenic neoplasm that has previously been shown to respond to Ab (Hodi et al., 2010). In MEL patients, significant ORR was observed with nivolumab at different overall rates of about 31% over different doses of 0.1, 0.3, 1, 3, or 10 mg / kg, as well as about 30% equivalent ORR was observed in RCC patients treated with 1 or 10 mg / kg nivolumab dose (see Example 7). In contrast, immunotherapy has historically had a very low success rate in lung cancer. This failure has been attributed to NSCLC being "non-immunogenic" (see, for example, Holt and Disis, 2008; Holt et al., 2011). The ability of lung cancer to block the immune system is due to a number of factors, including secretion of immunosuppressive cytokines, loss of major histocompatibility complex antigen expression, and co-opting of T cell inhibitory pathways ( Dasanu et al., 2012; Marincola et al., 2000; Brahmer et al., 2012).

  Despite the limited efficacy of immunotherapy historically found in lung cancer and the various mechanisms used by lung cancer cells to evade the immune system attack, various tumor cell vaccines (eg, Cells (belagenpumatucel) -L, whole cell-based vaccines that block the action of TGF-β2) and antigen-based vaccines that enhance antigen-specific anti-tumor immunity (eg, humoral EGF vaccines and MAGE-A3 fusion proteins or tumors) Vaccines that include some of the relevant MUC1 antigens) have been evaluated in clinical trials (Holt et al., 2011; Shepherd et al., 2011; Dasanu et al., 2012; Brahmer et al., 2012). However, while such antigen-specific vaccines have shown some promise for the treatment of NSCLC, Holt et al. (2011) suggest that testing for non-specific immunotherapeutic interventions improves results in NSCLC. Failed, suggesting the need to combine them with antigen-specific vaccines. These authors state the view that a truly effective immunotherapy of NSCLC is only due to the implementation of both strategies to enhance anti-tumor immunity and neutralize tumor-mediated immunosuppression, and the presence in NSCLC patients It was concluded that no or weak early immune response was unlikely that nonspecific stimulation of the immune system or elimination of immunosuppression would result in a change in clinical outcome.

  Thus, the results reported herein at NSCLC are particularly remarkable, unexpected and surprising. In NSCLC patients, OR was observed at 3%, 24% and 20% response rates at nivolumab doses of 1, 3 or 10 mg / kg, respectively (see Example 7). OR was observed through NSCLC tissues: 9 out of 54 squamous epithelia (17%) and 13 out of 74 non-squamous epithelia (18%). This level of activity seen with nivolumab in NSCLC patients with significant previous treatment (54% in 3 lines of previous treatment) and via tissues is unprecedented, especially in squamous tissue patients (Gridelli et al., 2008; Miller, 2006), which provides a very favorable benefit / risk dynamic in terms of efficacy and safety compared to existing standard treatments.

  In one aspect of the present immunotherapy, the anti-PD-1 Ab is administered to advanced or metastatic squamous or non-squamous NSCLC after platinum-based therapy (regardless of maintenance) and one other treatment. Shown locally as monotherapy. In other embodiments, the anti-PD-1 Ab is monotherapy locally in advanced or metastatic squamous or non-squamous NSCLC after treatment with platinum and one other previous chemotherapeutic regimen failure. Indicated as therapy. In a further aspect, the anti-PD-1 Ab is a single agent topically in advanced or metastatic squamous or non-squamous NSCLC after two lines of failure in one treatment that must include a platinum-based regimen Indicated as therapy. In yet a further aspect, the anti-PD-1 Ab is administered after at least one prior chemotherapy, after failure of at least one prior platinum-based treatment, or after progression to at least one platinum doublet treatment. It is indicated as a monotherapy topically in advanced or metastatic squamous or non-squamous NSCLC. In all of the immunotherapy methods described herein, treatment can be continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

Persistence of clinical response to anti-PD-1 in well-pretreated cancer patients The key role of the PD-1 pathway in suppressing antitumor immunity first demonstrated in experimental models is currently in clinical trials Has been confirmed. As described herein, monotherapy with a drug that blocks PD-1 (nivolumab) or its primary ligand, PD-L1 (BMS-936559), is a treatment-refractory progression Regression can be mediated in patients with sexual cancer. Negatively treated NSCLC patients receiving anti-PD-1 Ab, including patients with squamous epithelium, because standard salvage therapy has historically shown marginal benefit in these patients (Scagliotti et al., 2011) The objective response rate (ORR) in is surprising and unpredictable. The OR was prolonged with a median response period of about 18 months in 22 of 129 responders as measured by standard RECIST in this study (see Example 7). In addition, a pattern of tumor regression was observed consistent with the immune-related pattern of response.

  These findings have established the PD-1 pathway as a novel therapeutic focus in oncology (Pardoll, 2012; Topalian et al., 2012c; Hamid and Carvajal, 2013). In a current study in which 54% of patients had progressive disease after three or more previous systemic regimens, a preliminary analysis was performed by March 2013. This latest analysis supports and enhances the data and conclusions reached from the previous February 2012 analysis. Thus, conventional OR or long-term disease stabilization was observed in NSCLC (17% and 10%, respectively), MEL (31%, 7%) and RCC (29%, 27%) over all doses tested. Proven in patients (see Example 7). In addition, 13 patients (4%) exhibited an unusual “immune-related” response pattern as previously described with anti-CTLA-4 treatment, some of whom sustained (Sharma et al., 2011).

  The persistence of OR across multiple cancer types in patients treated with anti-PD-1 Ab is particularly significant. The latest analysis again indicates that some melanomas with progressive melanoma, which have not generally been observed to date with chemotherapy or small molecule inhibitors, but receive immunotherapy with ipilimumab and high doses of interleukin-2 We emphasized the persistence of clinical activity in nivolumab-treated patients that has been observed in patients (Topalian et al., 2011; Hodi et al., 2010). The persistence of partial tumor regression after drug discontinuation ultimately proves that PD-1 blockade resets the immune balance between tumor and host and OS benefit is significantly longer than previously measured Suggest that you can. Further follow-up is needed to determine the ultimate persistence of tumor regression and disease stabilization in patients for these clinical trials.

  Sustained objective tumor regression and disease stabilization induced by nivolumab in tightly pre-treated patients with strikingly progressive NSCLC, MEL and RCC is due to conventional chemotherapy and / or tyrosine-kinase inhibitors ( TKI) translates into a survival outcome that outweighs historical data for these patient populations treated with the treatment. In NSCLC, 1 and 2 year survival rates were 42% and 14%, respectively, with 9.2 and 10.1 months of central OS in patients with squamous and non-squamous cell carcinoma, respectively (Examples). 7). This high level of efficacy is particularly excellent because 54% of these patients have received three or more previous treatments. In addition, these figures may vary depending on data maturity, as the tracking of large numbers of lung cancer patients was relatively limited. Historically, 2L chemotherapy for lung cancer (ie, docetaxel and pemetrexed) has achieved a central OS of 7.5-8.3 months and a one-year survival rate of about 30% (Shepherd et al., 2000; Hanna et al., 2004). In the 2L / 3L population, erlotinib-treated patients had a median survival of 6.7 months versus 4.7 months in placebo-treated patients (Shepherd et al., 2005). Treatment is currently unapproved for use in lung cancer in settings above 3 L, with the exception of a retrospective review reporting a median survival of 5.8-6.5 months and a 25% one-year survival Thus, minimal data exists to assess survival in this patient population (Girard et al., 2009; Scartozi et al., 2010).

  In nivolumab-treated MEL patients, central OS at 16.8 months was achieved with breakthrough survival rates of 62% (1 year) and 43% (2 years) (see Example 7). Survival outcomes in pretreated melanoma patients supported the recent FDA approval of ipilimumab and vemurafenib. In a recent Phase 3 study involving melanoma patients with at least one previous treatment for metastatic disease, ipilimumab increased central OS from 6.4 to 10.1 months compared to gp100 peptide vaccine. (Hodi et al., 2010). In a phase 2 trial of ipilimumab in previously treated patients, 2-year survival rates ranged from 24.2-32.8% (Lebbe et al., 2012). Central OS in previously treated patients with BRAF mutant melanoma who participated in large phase 2 of vemurafenib was 15.9 months, with a one-year survival of 58% (Sosman et al., 2012).

  Thus, in one embodiment, immunotherapy with anti-PD-1 Ab is monotherapy locally or in advanced or metastatic MEL after treatment with dacarbazine (regardless of maintenance) and one other treatment Indicated as therapy. In other embodiments, the anti-PD-1 Ab is indicated as a monotherapy topically in advanced or metastatic MEL after failure of dacarbazine-based therapy. In a further aspect, the anti-PD-1 Ab is indicated as a monotherapy locally or in advanced or metastatic MEL after two lines of failure of one treatment that must include a dacarbazine-based regimen. In yet a further aspect, the anti-PD-1 Ab is an advanced or metastatic MEL after at least one prior chemotherapy, after at least one prior failure of dacarbazine-based treatment, or at least after progression to dacarbazine treatment. Locally indicated as monotherapy. In all of the immunotherapy methods described herein, treatment can be continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

In nivolumab-treated patients with RCC, central OS was reached at 22 months in 45% receiving 3 or more previous treatments and 71% receiving previous anti-angiogenic treatments (March 2013 analysis s Day). Breakthrough survival rates of 70% (1 year) and 50% (2 years) were observed (see Example 7). In a recent Phase 3 trial involving patients with renal cancer who progressed to disease after antiangiogenic therapy, everolimus was compared to placebo: central OS was 14.8 vs. 14.4 months, respectively (Motzer et al., 2008; Motzer et al., 2010). Recent Phase 3 trials comparing sorafenib and temsirolimus in the sunitinib refractory renal cancer population resulted in central OS at 16.6 and 12.3 months, respectively (Hutson et al., 2012). Thus, for NSCLC and MEL, treatment of a tightly pretreated RCC patient population with nivolumab resulted in a significantly longer central OS (> 22 months) than treatment of a poorly refractory population with standard therapy. Controlled phase 3 trials with promising survival endpoints are ongoing at NSCLC, MEL and RCC (NCT01673867, NCT01721772, NCT016642004, NCT01666884 and NCT017271746 (Clinical Trials Website, http: //www.clinicaltrials). .gov.) the results from these studies are expected to further demonstrate the high efficacy and persistence of responses to nivolumab in these cancers compared to standard therapy.

  In one embodiment, immunotherapy with anti-PD-1 Ab is localized to advanced or metastatic RCC following treatment with anti-angiogenic TKI or mTOR inhibitor (regardless of maintenance) and one other treatment. As monotherapy. In other embodiments, the anti-PD-1 Ab is indicated as a monotherapy topically in advanced or metastatic RCC following failure of treatment with an anti-angiogenic TKI or mTOR inhibitor. In a further aspect, the anti-PD-1 Ab is indicated as a monotherapy locally or in advanced or metastatic RCC after two lines of failure of one treatment that must include an anti-angiogenic TKI or mTOR inhibitor. It is. In a still further aspect, the anti-PD-1 Ab is administered after at least one prior chemotherapy, at least one prior failure of an anti-angiogenic TKI or mTOR inhibitor-based treatment, or at least an anti-angiogenic TKI or mTOR. After progression to inhibitor treatment, advanced or metastatic RCC is indicated as topical monotherapy. Anti-PD-1 immunotherapy may be continued as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

  Remarkably, OS in lung, melanoma and kidney cancer patients receiving nivolumab was significantly longer than PFS. These results mirror what has been reported for ipilimumab (Hodi et al., 2010), with early tumor growth or new lesion development in some patients undergoing immune checkpoint blockade leading to disease stabilization or regression. Reflects the observation that progress can be made. These findings suggest that progression-free survival may not be the optimal endpoint to determine the efficacy of nivolumab and other drugs in this class.

  The data described herein, demonstrating the high efficacy, longevity and broad applicability of anti-PD-1 immunotherapy for treating cancer, is based on nivolumab being tested against additional types of cancer Has been reached. For example, increased PD-L1 expression has been reported in various hematologic malignancies and based on the ability to prevent a host immune response from having a beneficial effect on malignant cells, based on hematologic malignancies (multiple myeloma). , B cell lymphoma, T cell lymphoma, Hodgkin lymphoma / primary mediastinal B cell lymphoma, and chronic myelogenous leukemia) have been initiated to confirm the ability of nivolumab to mediate antitumor activity (NCT 0159370). ). Nivolumab is also tested as a monotherapy in advanced hepatocellular carcinoma (NCT01658878).

  In summary, the results of the anti-PD-1 immunotherapy described herein are significant in at least three ways: First, the efficacy of anti-PD-1 has been shown to outweigh historical efficacy data for patients in standard therapy treatments for cancer. Remarkably, this efficacy has been demonstrated in patients in a well-pretreated population where about half of the patients had progressive disease after three or more previous systemic regimens. Such patients suffering from advanced, metastatic and / or refractory cancer are notoriously difficult to treat. Accordingly, the present application is a method for immunotherapy of a patient suffering from advanced, metastatic and / or refractory cancer, comprising the interaction of PD-1 with PD-L1 and / or PD-L2. A method comprising administering to a patient a therapeutically effective amount of an Ab or antigen-binding portion thereof that disrupts the effect. In any one of the aspects of the therapeutic methods described herein, the subject has been pre-treated for cancer; for example, the subject has at least one, two, or three treatments for cancer. Received the previous line.

  Second, it has been shown that the treatment methods of the present application can be applied to a wide variety of cancers in a broad genus. Even “non-immunogenic” cancers, such as NSCLC (Holt et al., 2011) and cancers that are difficult to treat, such as ovarian and gastric cancer (and other treated cancers, including MEL, RCC, and CRC) Based on the surprising finding that it can be applied to treatment with -PD-1 and / or anti-PD-L1 (see Examples 7 and 14), this application Provides a method for immunotherapy of a patient substantially afflicted with any of a wide range of cancers.

  Third, treatment with anti-PD-1 or anti-PD-L1 Ab has been shown to result in significantly lasting clinical activity in cancer patients. Accordingly, the present application provides a method for sustained release in a cancer patient comprising administering to the patient a therapeutically effective amount of an Ab or antigen-binding portion thereof that disrupts the interaction of PD-1 with PD-L1 and / or PD-L2. To provide an immunotherapeutic method for inducing a novel clinical response. In a preferred embodiment of any of the treatment methods described herein, the clinical response is a sustained response.

  As used herein, a “sustained” response is a treatment or clinical response that exceeds the expected median OS rate in a patient population. The expected median OS rate varies for different cancers and different patient populations. In one embodiment, the sustained response exceeds the expected median OS rate in the relevant patient population by at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 50%. The main benefit of an immunotherapeutic approach based on PD-1 pathway blockage is that memory T cells that can maintain anti-tumor immune surveillance and inhibit tumor growth for extended periods of time, even in the absence of continuous therapy, for many years It can be a function recovery of exhausted T cells in long-term production of (Kim and Ahmed, 2010). Indeed, long-term follow-up studies on patients after discontinuation of nivolumab treatment have shown that patients with CRC have experienced a complete response that had continued after 3 years; patients with RCC had sustained 3 years of no treatment. A partial response that has been converted to a complete response that lasted in December; and that the patient with melanoma achieved a partial response that was stable for 16 months without treatment, And recurrent disease has been successfully treated with redirected anti-PD-1 therapy (Lipson et al., 2013).

Immune-Related Clinical Reactions It has been shown that conventional response criteria cannot adequately assess the activity of immunotherapeutic agents because progressive disease (by initial radiological assessment) does not always reflect therapeutic failure. For example, treatment with the anti-CTLA-4 Ab, ipilimumab, has been shown to produce four distinct response patterns, all of which were associated with good survival: (a) without new lesions Reduction in baseline lesions; (b) persistent stable disease (in some patients, then a slow and steady decrease in total tumor burden); (c) post-increase response in total tumor burden; And (d) Response in the presence of new lesions. Therefore, in order to properly evaluate an immunotherapeutic, the long-term effects on the target disease must also be captured. In this regard, a systematic immune-related response criterion (irRC) has been proposed that seeks to enhance the characterization of immune-related response patterns, taking into account the initial increase in tumor burden and / or the development of new lesions. (Wolchok et al., 2009). Although the full impact of these unconventional response patterns is still not revealed in a randomized trial of nivolumab on survival endpoints, this observation was found with ipilimumab where a significant prolongation of OS was observed in treated patients (Hodi et al., 2010; Robert et al., 2011).

  The overall risk / benefit profile of anti-PD-1 immunotherapy is also a more severe drug-related adverse event (AE;> grade 3), a particular event hitherto consistently observed with other immunotherapy agents. ), Which is preferred. This suggests that anti-PD-1 immunotherapy can be delivered in an outpatient setting with minimal symptomatic treatment.

Broad Range of Cancers Treatable by Anti-PD-1 Immunotherapy The clinical data presented herein suggests that immunotherapy based on PD-1 blockade may only affect "immunogenic" tumor types, such as MEL and RCC Demonstrates that it extends to tumor types not generally considered to be immune responses, including but not limited to NSCLC. The unexpected success with treatment-refractory metastatic NSCLC highlights the possibility that any neoplasm may be "immunogenic" in the context of appropriate immunomodulation, and PD-1 blockade as an immunotherapeutic approach is highly variable. Suggest broad application across a range of tumor types. Thus, cancers that can be treated using the anti-PD-1 Abs of the present invention also include those that generally respond to immunotherapy as well as those that are conventionally considered non-immunogenic. Non-limiting examples of preferred cancers for treatment include NSCLC, MEL, RCC, CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, esophagus, ovary, gastrointestinal and breast carcinomas, and hematologic malignancies . Although NSCLC is not considered to be generally responsive to immunotherapy, the data described herein unexpectedly indicate that both squamous and non-squamous NSCLC were treated with anti-PD-1 Ab. Prove to respond to Further, the application provides for the treatment of refractory or recurrent malignancies whose growth may be inhibited using the anti-PD-1 Ab of the invention.

  Based on the indications provided by the very broad applicability of anti-PD-1 immunotherapy provided herein, examples of cancers that can be treated using anti-PD-1 Abs in the methods of the invention include: Liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, skin or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colorectal cancer, transrectal cancer, anus Area cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system Cancer of the thyroid, cancer of the thyroid, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, cancer of the urethra, cancer of the penis, pediatric solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis Carcinoma, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis Tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environment-induced cancers including asbestos-induced cancers, hematological malignancies such as multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / Primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large Cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and above Includes any combination of cancers. The invention is also applicable to the treatment of metastatic cancer.

Medical uses of anti-PD-1 Abs One aspect of the present invention inhibits signaling from the PD-1 / PD-L1 pathway and thereby enhances the endogenous immune response in subjects with cancer The use of any of the anti-PD-1 Abs or antigen-binding portions thereof of the present invention for the manufacture of a medicament to Another aspect is the use of any of the anti-inflammatory agents of the present invention for the manufacture of a medicament for immunotherapy of a subject suffering from cancer, including disrupting the interaction between PD-1 and PD-L1. Use of PD-1 Ab or an antigen binding portion thereof. These uses for the manufacture of a medicament are broadly applicable to all the ranges of cancer described herein. In a preferred embodiment of these uses, the cancer is squamous NSCLC, non-squamous NSCLC, MEL, RCC, CRC, CRPC, HCC, squamous cell carcinoma of the head and neck, and carcinoma of the esophagus, ovary, gastrointestinal tract and breast, And hematological malignancies. The present application also relates to the medicine of any anti-PD-1 Ab or antigen-binding portion thereof of the invention corresponding to all aspects of the method of treatment using an anti-PD-1 Ab described herein. To provide strategic use.

  The present application also includes for use in treating a subject suffering from cancer, including enhancing an endogenous immune response in the subject by inhibiting signaling from the PD-1 / PD-L1 pathway. , An anti-PD-1 Ab of the invention or an antigen binding portion thereof. The present application provides an anti-PD-1 Ab or antigen thereof of the invention for use in immunotherapy of a subject with cancer, comprising disrupting the interaction between PD-1 and PD-L1. A binding moiety is further provided. These Abs can be used in enhancing the endogenous immune response to all of the cancers described herein, or in immunotherapy of all of the cancers described herein. . In preferred embodiments, the cancer is squamous NSCLC, non-squamous NSCLC, MEL (eg, metastatic malignant MEL), RCC, CRC, CRPC, HCC, head and neck squamous cell carcinoma, and esophagus, ovary, gastrointestinal tract and Includes breast carcinoma, and hematological malignancies.

Combination Therapy Containing Anti-PD-1 Antibodies Monotherapy with anti-PD-1 and anti-PD-L1 Abs may improve the survival of patients with lung cancer, melanoma, kidney cancer, and other possible malignancies Although shown to increase significantly, preclinical data indicate that synergistic treatment combinations based on PD-1 pathway blockage can have even more potent effects. Clinical mechanisms of nivolumab combined with ipilimumab (anti-CTLA-4) continue to be similar in mechanism of action to nivolumab so far (Parry et al., 2005; Mellman et al., 2011; Topalian et al., 2012c) And the results from Phase 1 studies are provided herein (NCT010242231; NCT018445505; NCT01783938; Wolchok et al., 2013a; Wolchok et al., 2013b; Hodi et al., 2013). Clinical trials have also demonstrated the administration of nivolumab in patients with advanced solid tumors with melanoma vaccines (NCT01176461, NCT01176474; Weber et al., 2013), ipilimumab (BMS-986015), human IgG4 anti-KIR Ab and (NCT01714739; Sanborn). Et al., 2013), in patients with advanced or metastatic solid tumors with cytokines such as IL-21 (NCT01629758; Chow et al., 2013), dual chemotherapy with chemotherapeutic drugs such as platinum and naive chemotherapy. Therapy has been initiated in patients with NSCLC (NCT01454102; Rizvi et al., 2013) and in patients with small molecule targeted therapy and metastatic RCC (NCT01472881; Amin et al., 2013).

  In one aspect, the application relates to the combination of an anti-PD-1 Ab with different cancer treatments for the treatment of various cancers, including chemotherapeutic regimens, radiation therapy, surgery, hormone loss and angiogenesis inhibitors. About. PD-1 blockade also involves immunogens, eg, preparations of cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), antigen-presenting cells, eg, trees with tumor-associated antigens. Dendritic cells, cells transfected with a gene encoding an immunostimulatory cytokine (He et al., 2004), and / or another immunotherapeutic Ab (e.g., anti-CTLA-4, anti-PD-L1 and / or anti- It can be effectively combined with LAG-3 Ab). Non-limiting examples of tumor vaccines that can be used include those that express a peptide of a melanoma antigen, such as a peptide of gp100, a MAGE antigen, Trp-2, MART1 and / or tyrosinase, or the cytokine GM-CSF. Includes transfected tumor cells.

Combination of anti-PD-1 and anti-CTLA-4 Ab to treat advanced melanoma Non-redundant immunological checkpoints, T-cell activation, proliferation and effector function and / or function within lymph nodes Given that the combination of anti-CTLA-4 and anti-PD-1 had a stronger anti-tumor effect in a mouse tumor model than either Ab alone, given that it could inhibit the intratumoral microenvironment. Based on clinical data (see US Pat. No. 8,008,449), the hypothesis that combined blockade of CTLA-4 and PD-1 can produce better antitumor activity than single agents has been hypothesized in MEL patients. Tested in a clinical trial (Example 15).

  Although not formally compared in this study, the concurrent nivolumab / ipilimumab regimen achieved an ORR in excess of that achieved with either nivolumab (Example 7) or ipilimumab alone (Hodi et al., 2010). Very importantly, a rapid and deep response is achieved in a significant proportion of treated patients, a "deep" tumor response being a target characterized by a reduction of more than 80% from baseline by radiological assessment 3 shows the response at the lesion. In this study, the majority of responding patients, including those with extensive and massive tumor burden, achieved> 80% tumor regression at initial tumor assessment. Particularly impressive are the simultaneous regimens ((i) the combined administration of anti-PD-1 and anti-CTLA-4 Ab, followed by the induction dosing schedule with administration of anti-PD-1 Ab alone, and (ii) ) (Including a maintenance dosing schedule that includes infrequent combination administration of anti-PD-1 and anti-CTLA-4 Ab) with a 31% evaluable response by> 12 weeks by> 80% tumor The observation was evidence of regression. At MTD for the concurrent regimen, all 9 responders demonstrated> 80% tumor regression with 3 CRs. In contrast, <3% of MEL patients who have received nivolumab or ipilimumab at 3 mg / kg in the clinical experience to date have achieved CR (Example 7; Hodi et al., 2010). Thus, the overall activity of this immunotherapy combination in this preliminary phase 1 study is almost as compared to other drugs approved or developed for advanced melanoma, including targeting inhibitors of activated kinases. There is no inferiority (Chapman et al., 2011). A further advantage of this combination is the persistence of the response as demonstrated in this study with nivolumab (as described herein) and ipilimumab as well as in long-term immunotherapy trials (Wolchok et al., 2013d).

  These initial data suggest that a faster response of better magnitude can be achieved in patients treated with the nivolumab / ipilimumab combination compared to the historical experience of either drug alone. Responses were generally persistent and were observed even in patients where treatment was terminated early due to toxicity. Responders included patients with elevated LDH, M1c disease and massive multiple tumor burden. Similar to previous reports on ipilimumab or nivolumab monotherapy, conventional ORRs have shown that nivolumab / ipilim in that many patients experienced either long-term SD or immune-related patterns that are unconventional to response. The range of clinical activity and potential benefits in patients treated with bleedlimen concomitantly cannot be fully captured. Indeed, of the seven patients in the concurrent regimen with SD ≧ 24 weeks or irSD ≧ 24 weeks as the best OR, six demonstrated at least 19% significant tumor regression, and the seventh patient after long term SD Has reduced tumor burden. Previous experience with checkpoint-blocking monotherapy supports the observation that some patients can survive long-term with SD as the best OR, suggesting that restoring the equilibrium phase of immune surveillance is a desirable outcome. Bring evidence (Screiber et al., 2011).

  When patients are treated sequentially with nivolumab after previous ipilimumab, the observations that can achieve OR indicate that the unresponsiveness to CTLA-4 blockade does not impair the clinical benefit from PD-1 blockade. 4 further supports the non-redundant nature of the co-inhibition pathway of. Strikingly, the data described herein (Example 8) suggests an association between the development of a response and tumor PD-L1 expression in patients receiving nivolumab, while previous data was treated with ipilimumab Figure 4 shows a correlation between OS and increased peripheral ALC in patients (Berman et al., 2009; Ku et al., 2010; Postow et al., 2012; Delyon et al., 2013). In this study of the nivolumab / ipilimumab combination, a clinical response was observed in patients irrespective of lymphocyte counts or baseline tumor PD-L1 expression (Example 17), and the immune response generated by the combination therapy was either single. It suggested that it had unique properties compared to drug therapy. The data suggest that baseline tumor PD-L1 expression and lymphocyte counts may be less relevant for setting an active combination regimen that can induce rapid and significant tumor regression. However, and notably, different anti-PD-L1 Abs (rabbit mAb28-8 vs. mouse 5H1 mAb) were used to measure PD-L1 expression in combination therapy studies as compared to nivolumab monotherapy studies. Used in different IHC assays. In addition to changes in IHC assays and Abs, different results may also reflect differences in biopsy samples and tumor heterogeneity. The utility of PD-L1 expression as a biomarker for anti-PD-1 efficacy is further evaluated in randomized phase 3 trials (see, eg, NCT01721772, NCT01666884, and NCT017271746).

  The range of adverse events observed in patients treated with the concomitant regimen was qualitatively similar to experience with nivolumab or ipilimumab monotherapy, and the proportion of AEs was increased in patients treated with the combination . Grade 3-4 treatment-related AEs were 20% in patients treated with ipilimumab monotherapy at a dose of 3 mg / kg (Hodi et al., 2010) and 17% in patients treated with nivolumab alone (Example Compared to the historical ratio of 5), it was observed in 53% of patients treated with the concurrent regimen of nivolumab / ipilimumab. In the continuous regimen cohort, 18% of patients experienced grade 3-4 treatment-related AEs. AEs experienced by patients treated with simultaneous and sequential regimens were manageable and / or generally reversible using existing treatment algorithms.

  Collectively, these results suggest that nivolumab and ipilimumab can be administered simultaneously with a manageable safety profile, causing a sustained clinical response. A faster and deeper clinical tumor response was observed in patients treated with the combination as compared to the response obtained with either single agent.

  On the clinical cut-off date of February 2013 for the study described in Example 15, of the 52 subjects in the concurrent regimen that could be assessed for response, 21 (40%) had modified World Health Had an OR by institutional (mWHO) criteria (Wolchok et al., 2009). In two additional subjects (4%), there was an unconfirmed OR. In cohort 1 (0.3 mg / kg nivolumab and 3 mg / kg ipilimumab), 3 out of 14 evaluable subjects had OR with mWHO (ORR: 21%, 1 CR and 2 PR). In cohort 2 (1 mg / kg nivolumab and 3 mg / kg ipilimumab), 9 out of 17 evaluable subjects had an OR with mWHO (ORR: 53%; 3 CR and 6 PR) including). In cohort 2a (3 mg / kg nivolumab and 1 mg / kg ipilimumab), 6 out of 15 evaluable subjects had OR with mWHO (ORR: 40%; 1 CR and 5 PR) including). In cohort 3 (3 mg / kg nivolumab and 3 mg / kg ipilimumab), 3 out of 6 evaluable subjects had an objective response by mWHO (ORR: 50%, including 3 PRs) . Based on these data, the invention described herein provides (a) an Ab or antigen-binding portion thereof that specifically binds and inhibits PD-1; and (b) specificity for CTLA-4 A method of treating a subject suffering from cancer comprising administering to a subject an Ab or an antigen-binding portion thereof that specifically binds and inhibits, wherein each Ab is from 0.1 to 20. 0 mg / kg body weight, wherein the concurrent regimen comprises (i) anti-PD-1 and anti-PD-1 at a dose frequency of at least once every two, three or four weeks, or at least once a month. Administering the combination of CTLA-4 Abs at least 2, 4, 6, 8 or 10 times, and then administering the anti-CLA-4 Ab at least once every 2, 3 or 4 weeks or at least once a month. PD-1 Ab alone An inducible dosing schedule comprising administering at least 2, 4, 6, 8 or 12 times; then (ii) at a frequency of at least once every 8, 12 or 16 weeks, or at least once a quarter, or clinically. At least 4, 6, 8, 10, 12, or 16 doses of the combination of anti-PD-1 and anti-CTLA-4 Ab for as long as therapeutic benefit is observed or until unmanageable toxicity or disease progression occurs Including a maintenance dosing schedule that includes the steps of:

  In one embodiment of this method, the maintenance dosing schedule comprises administering the combination of anti-PD-1 and anti-CTLA-4 Ab up to 4, 6, 8, 10, 12, or 16 times. In other embodiments, the concurrent regimen comprises: (i) administering a combination of anti-PD-1 and anti-CTLA-4 Ab to 2, 2, 3 or 4 weeks, or once a month; 4, 6, or 8 doses and then once every 2, 3 or 4 weeks, or once a month, the anti-PD-1 Ab alone, 2, 4, 6, 8 or Induction dosing schedule including 12 doses; then (ii) once every 8, 12 or 16 weeks, or once every quarter, or as long as clinical benefit is observed, or A maintenance dosing schedule comprising administering 4, 6, 8, 10, 12, or 16 doses of the combination of anti-PD-1 and anti-CTLA-4 Ab until disease progression occurs. In one other embodiment, each anti-PD-1 and anti-CTLA-4 Ab is individually 0.1, 0.3, 0.5, 1, 3, 5, 10, or 20 mg / kg. Administered in doses. In a further aspect, the dose of each anti-PD-1 and anti-CTLA-4 Ab is kept constant during the induction and maintenance dosing schedules. In yet other embodiments, the anti-PD-1 and anti-CTLA-4 Ab are administered at the following doses: (a) 0.1 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab (B) 0.3 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab; (c) 1 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA- 4 Ab; (d) 3 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab; (e) 5 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA- 4 Ab; (f) 10 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab; (g) 0.1 mg / kg anti-PD-1 Ab and 1 mg / kg anti- CTLA-4 Ab; (h) 0 3 mg / kg anti-PD-1 Ab and 1 mg / kg anti-CTLA-4 Ab; (i) 1 mg / kg anti-PD-1 Ab and 1 mg / kg anti-CTLA-4 Ab; (j) (K) 5 mg / kg anti-PD-1 Ab and 1 mg / kg anti-CTLA-4 Ab; or (l) 3 mg / kg anti-PD-1 Ab and 1 mg / kg anti-CTLA-4 Ab; ) Administered at 10 mg / kg anti-PD-1 Ab and 1 mg / kg anti-CTLA-4 Ab.

  In the protocol described in Example 15, the dosing regimen of 3 mg / kg nivolumab and 3 mg / kg ipilimumab exceeds the MTD (the combination of ipilimumab and other anti-PD-1 Abs is higher or lower). Both cohort 2 (1 mg / kg nivolumab and 3 mg / kg ipilimumab) and cohort 2a (3 mg / kg nivolumab and 1 mg / kg ipilimumab) had similar clinical activity (although they may have MTD). . In addition, the majority of responses to the combination of nivolumab and ipilimumab occurred in the first 12 weeks. The uncertainty of whether ipilimumab given for 12 weeks contributes to clinical benefit and the schedule approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for ipilimumab for 3 weeks In a preferred embodiment, the anti-CTLA-4 Ab is administered during the induction dosing schedule once every three weeks, for a total of four doses, taking into account the fact that there is a total of four doses. Monotherapy treatment with nivolumab at 3 mg / kg every two weeks until progression has been shown to be associated with a sustained response (Examples 4-7), including maintenance including nivolumab every 12 weeks The dosing schedule has been shown to be effective (Example 15). Thus, starting at week 12 after completion of the four combined nivolumab and ipilimumab, nivolumab at 3 mg / kg can be administered every 2 to at least every 12 weeks until progression. Thus, in a preferred embodiment of the simultaneous regimen method, the anti-PD-1 and anti-CTLA-4 Ab comprise: (a) 1 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab; (B) Administered at doses of 3 mg / kg anti-PD-1 Ab and 1 mg / kg anti-CTLA-4 Ab, the concurrent regimen comprising (i) anti-PD with a dosing frequency of once every three weeks. Induced dosing schedule comprising administering the combination of -1 and anti-CTLA-4 Ab four times, then once every three weeks, administering anti-PD-1 alone four times; (Ii) anti-PD-1 and anti-PD-1 at a dosing frequency of 2 to 12 or more times per week or until clinical benefit is observed or until unmanageable toxicity or disease progression occurs. Of CTLA-4 antibody The combined further comprising a maintenance dosing schedule comprises administering up to 8 times.

  Exposure response analysis of nivolumab monotherapy over a dose range of 1 mg / kg to 10 mg / kg shows similar clinical activity (Example 7), but 0.3 mg / kg, 3 mg / kg and 10 mg / kg of ipilimumab alone Exposure response analysis of drug therapy has demonstrated that activity increases with increasing dose in phase 2 trials (Wolchok et al., 2010). Therefore, the dose of 3 mg / kg ipilimumab (Cohort 2) may be more clinically influential than the choice of 3 mg / kg nivolumab (Cohort 2a). Thus, in a more preferred embodiment of the simultaneous regimen method, the anti-PD-1 and anti-CTLA-4 Ab are administered at a dose of 1 mg / kg anti-PD-1 Ab and 3 mg / kg anti-CTLA-4 Ab. Is done.

  In one embodiment of the present method, the anti-PD-1 and anti-CTLA-4 Ab are formulated for intravenous administration. In one other embodiment, when anti-PD-1 and anti-CTLA-4 Ab are administered in combination, they are administered within 30 minutes of each other. Any of the Abs may be administered first, ie, in one embodiment, the anti-PD-1 Ab is administered before the anti-CTLA-4 Ab, while in other embodiments, the anti-CTLA-4 Ab Is administered before the anti-PD-1 Ab. Generally, any of the Abs will be administered intravenously over a period of 60 minutes. In a further aspect, the anti-PD-1 and anti-CTLA-4 Ab are mixed together as a single composition in a pharmaceutically acceptable formulation for simultaneous administration, or a pharmaceutically acceptable formulation. At the same time as any of the Abs in a separate composition.

  The data described in Example 7 demonstrates that immunotherapy with nivolumab resulted in significant clinical activity in MEL patients who did not respond to previous ipilimumab treatment. Accordingly, the present application is a continuous regimen method for treating a subject suffering from cancer, previously treated with an anti-CTLA-4 Ab, comprising from 0.1 to 20.0 mg / kg body weight. At doses in the range and at least once a week, at least once every two, three or four weeks, or at least once a month, or as long as clinical benefit is observed or difficult to manage Provided is a method comprising administering to a subject up to 6 to 72 times an Ab or antigen binding portion thereof that specifically binds and inhibits PD-1 until toxicity or disease progression occurs. In one embodiment of this method, administration of the anti-PD-1 Ab to the subject is initiated within 1-24 weeks after the last treatment with the anti-CTLA-4 Ab. In other embodiments, administration of the anti-PD-1 Ab to the subject is initiated within 1, 2, 4, 8, 12, 16, 20, or 24 weeks after the last treatment with the anti-CTLA-4 Ab. You. In a preferred embodiment, administration of the anti-PD-1 Ab is initiated within 4, 8, or 12 weeks after the subject's last treatment with the anti-CTLA-4 Ab. One embodiment of the method comprises administering the anti-PD-1 Ab at a dose of 0.1-20 mg / kg, for example 0.1, 0.3, 0.5, 1, 3, 5, 10, or 20 mg / kg. Administering to the subject. In a preferred embodiment, the anti-PD-1 Ab is administered at a dose of 1 or 3 mg / kg. In one embodiment, a continuous regimen is administered once a week, once every 2, 3 or 4 weeks, or once a month, or as long as clinical benefit is observed, or is difficult to manage. Including 6-72 doses of anti-PD-1 Ab to the subject until toxicity or disease progression occurs. In a preferred embodiment, anti-PD-1 is administered at a dose of 1 or 3 mg / kg up to 48 times, with a dose frequency of once every two weeks. In another preferred embodiment, the anti-PD-1 Ab is formulated for intravenous administration.

  In one aspect of any of the simultaneous or sequential regimen methods of the present application, the treatment comprises reducing tumor size and / or growth, removing the tumor, reducing the number of metastatic lesions over time, complete response, partial response, and Produces at least one therapeutic effect selected from stable diseases. Based on the wide range of cancers to which nivolumab has shown clinical response, the combination therapy method of the present application is also applicable to a variety of cancers. Examples of cancers that can be treated by these methods include liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, skin or intraocular melanoma, kidney cancer, uterine cancer, ovarian cancer, Colorectal cancer, colorectal cancer, transrectal cancer, cancer of the anal area, gastric cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, non-Hodgkin Lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, thyroid cancer, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcoma, urethra cancer, penis cancer, pediatric solid tumor, bladder cancer, kidney or Ureteral carcinoma, renal pelvic carcinoma, CNS neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, Environment-induced cancers, including asbestos-induced cancers, hematological malignancies, such as multiple myeloma, Cell lymphoma, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, bar Kit lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T cell lymphoma, and precursor T-lymphoma Includes lymphoblastic lymphoma, and any combination of the above cancers. The invention is also applicable to the treatment of metastatic, refractory or recurrent cancer. In a preferred embodiment, the cancer to be treated is MEL, RCC, squamous NSCLC, non-squamous NSCLC, CRC, CRPC, OV, GC, HCC, PC, squamous cell carcinoma of the head and neck, esophagus, gastrointestinal tract and breast carcinoma , And hematological malignancies. In a more preferred embodiment, the cancer is MEL.

  In one embodiment, the subject has been pre-treated for cancer. For example, the patient may have been treated with one or more prior systemic regimens of the type described herein as standard therapy. In one other embodiment, the cancer is an advanced, recurrent, metastatic and / or refractory cancer. In a preferred embodiment, the concurrent or sequential regimen treatment induces a sustained clinical response in the subject. In a preferred embodiment, the subject is a human, wherein the anti-PD-1 Ab inhibits human PD-1 and the anti-CTLA-4 Ab inhibits human CTLA-4.

  The anti-PD-1 Ab used in the method of the present application can be any of the therapeutic anti-PD-1 Abs of the present invention. In a preferred embodiment, the anti-PD-1 Ab is a mAb, which can be chimeric, humanized or human. In one embodiment, the anti-PD-1 Ab is the 17D8, 2D3, 4H1, 5C4 (nivolumab), 4A11, 7D3 or 5F4, respectively, described and characterized in U.S. Patent No. 8,008,449. Includes CDR1, CDR2 and CDR3 domains in the heavy chain variable region and CDR1, CDR2 and CDR3 domains in the light chain variable region. In a further aspect, the anti-PD-1 Ab comprises the heavy and light chain variable regions of each of 17D8, 2D3, 4H1, 5C4 (nivolumab), 4A11, 7D3, or 5F4. In a further aspect, the anti-PD-1 Ab is 17D8, 2D3, 4H1, 5C4 (nivolumab), 4A11, 7D3 or 5F4. In a preferred embodiment, the anti-PD-1 Ab is nivolumab.

  The anti-CTLA-4 antibodies of the invention bind to human CTLA-4 so as to disrupt the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 transduces a signal that causes the inactivation of T cells bearing CTLA-4 receptors, disruption of the interaction effectively activates such T cells. Induce, increase or prolong, thereby inducing, increasing or prolonging an immune response. Anti-CTLA-4 Abs are described, for example, in U.S. Patent Nos. 6,051,227, 7,034,121 in PCT Application Publications WO 00/37504 and WO 01/14424. A typical clinical anti-CTLA-4 Ab is a human mAb 10D1 (now known as ipilimumab and marketed as YERVOY®) as described in US Pat. No. 6,984,720. ). In one aspect of any of the methods of the present application, the anti-CTLA-4 Ab is a mAb. In one other embodiment, the anti-CTLA-4 antibody is a chimeric, humanized or human antibody. In a preferred embodiment, the anti-CTLA-4 antibody is ipilimumab.

  The present application also provides for the manufacture of a co-administered medicament for treating a subject suffering from cancer in a concurrent regimen, the anti-PD-A in combination with an anti-CTLA-4 Ab or antigen binding portion thereof. The use of 1 Ab or an antigen-binding portion thereof, wherein the anti-PD-1 and anti-CTLA-4 Ab are each administered at a dose ranging from 0.1 to 20.0 mg / kg body weight, and further comprising administering a simultaneous regimen. (I) administering a combination of anti-PD-1 and anti-CTLA-4 Ab at least once every 2, 3 or 4 weeks, or at least once a month, for at least 2, 4, 6, 8 or 12 doses, then the anti-PD-1 Ab alone at least once every 2, 3 or 4 weeks, or at least once a month. Or 10 times An induction dosing schedule comprising administering; then (ii) at least once every 8, 12, or 16 weeks, or at least once every quarter, or as long as clinical benefit is observed, or is difficult to manage Use comprising a maintenance dosing schedule comprising up to 4, 6, 8, 10, 12, or 16 doses of the combination of anti-PD-1 and anti-CTLA-4 Ab until toxicity or disease progression occurs. provide. The present application provides anti-PD-1 and anti-CTLA-4 Abs for the manufacture of co-administered medicaments corresponding to all aspects of the methods of treatment using these Abs described herein. And is broadly applicable to all ranges of cancer described herein.

  The present application also provides an anti-PD-1 Ab or antigen-binding portion thereof for use in combination with an anti-CTLA-4 Ab or antigen-binding portion thereof for treating a subject suffering from cancer in a concurrent regimen. Wherein the anti-PD-1 and anti-CTLA-4 Ab are each administered at a dose ranging from 0.1 to 20.0 mg / kg body weight, and wherein the concurrent regimen comprises (i) at least 2,3 Or at least two, four, six, eight or twelve doses of the combination of anti-PD-1 and anti-CTLA-4 antibodies at a dose frequency of once every four weeks, or at least once a month; Induced administration comprising administering anti-PD-1 alone at least 2, 4, 6, 8 or 10 times at a frequency of at least once every two, three or four weeks, or at least once a month Schedule Then (ii) at least once every 8, 12, or 16 weeks, or at least once every quarter, at a frequency of administration, or as long as clinical benefit is observed, or develop unmanageable toxicity or disease progression Anti-PD-1 Ab or antigen binding thereof, including a maintenance dosing schedule comprising up to 4, 6, 8, 10, 12, or 16 doses of a combination of anti-PD-1 and anti-CTLA-4 antibodies until Provide the part.

  Based on the PD-L1 biomarker assay, screen patient populations and select patients as appropriate for immunotherapy with a combination of anti-PD-1 and anti-CTLA-4 using a simultaneous or sequential regimen And methods for predicting the efficacy of Ab combinations, to the extent that this biomarker is applicable to combination therapy with anti-PD-1 and anti-CTLA-4 Abs, anti-PD-1 monotherapy It is performed as described for the therapy.

  This application relates to (a) Ab or an antigen-binding portion thereof that specifically binds and inhibits PD-1 at a dose ranging from 0.1 to 20.0 mg / kg body weight; An Ab or antigen-binding portion thereof that specifically binds and inhibits CTLA-4 at a dose in the range of 0.0 mg / kg; and (c) anti-PD-1 and anti-CTLA- Further provided is a kit for treating a subject afflicted with cancer, the kit comprising instructions for using the four antibody combination. In one embodiment, several doses of anti-PD-1 and anti-CTLA-4 Ab are mixed within a single pharmaceutical formulation for simultaneous administration. In other embodiments, the dose of anti-PD-1 and anti-CTLA-4 Ab is formulated as a separate composition having any of the Abs in a pharmaceutically acceptable formulation.

  This application relates to (a) Ab or an antigen-binding portion thereof that specifically binds and inhibits PD-1 at a dose ranging from 0.1 to 20.0 mg / kg body weight; and (b) a continuous regimen method. Further provided is a kit for treating a subject afflicted with cancer, comprising instructions for using the anti-PD-1 Ab in any of the cases.

  Combination PD-1 and CTLA-4 blockade can also be further combined with standard cancer treatments. For example, the combination PD-1 and CTLA-4 blockade can be effectively combined with a chemotherapeutic regimen for treatment of MEL, for example, a further combination with dacarbazine or IL-2. In these instances, it may be possible to reduce the dose of the chemotherapeutic agent. The scientific rationale is that, following the combined use of PD-1 and CTLA-4 blockade and chemotherapy, cell death as a result of the cytotoxic effects of many chemotherapeutic compounds resulted in increased levels of tumor antigens in the antigen presentation pathway. Is to bring. Other combination therapies that can produce a synergistic effect with combination PD-1 and CTLA-4 blockade through cell death include radiation therapy, surgery, or hormone loss. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with the combination PD-1 and CTLA-4 blockade. Inhibition of angiogenesis results in tumor cell death, which may also be a source of tumor antigens that enter the host antigen presentation pathway.

Immunotherapy of cancer patients using anti-PD-L1 antibody PD-L1 is a solid capable of inhibiting the cytokine production and cytolytic activity of PD-1-positive, tumor infiltrating CD4 ⁺ and CD8 ⁺ T cells, respectively. It is the major PD-1 ligand up-regulated in tumors (Dong et al., 2002; Hino et al., 2010; Taube et al., 2012). These properties make PD-L1 a promising target for cancer immunotherapy. Clinical trials of the anti-PD-L1 immunotherapy described in the Examples show that mAb blockade of the immunoinhibitory ligand PD-L1 includes metastatic NSCLC, MEL, including patients with a wide range of previous treatments. It demonstrates for the first time that it results in both sustained tumor regression and long-term (≧ 24 weeks) disease stabilization in patients with RCC and OV. The human anti-PD-L1 HuMAb, BMS-936559, has a favorable safety profile across doses below 10 mg / kg, as evidenced by the low (9%) incidence of grade 3-4 drug-related AEs. did. These headings things are, PD-L1 ^{- / -} mild autoimmune phenotype observed in mice (Dong et al., 2004) and ^{PD-1 - / -} as compared to the mouse, ^{CTLA-4 - / -} mice Is consistent with the more severe hyperproliferation seen in Phan et al., 2003; Tivol et al., 1995; Nishimura et al., 1999. Many of the toxicities associated with anti-PD-L1 administration in patients were immune-related, suggesting a qualifying effect. The range and frequency of adverse events of particular interest (AEOSI) vary slightly between anti-PD-L1 and anti-CTLA-4, highlighting the different biology of these pathways (Ribas et al., 2005). . Infusion reactions were observed with BMS-936559, but they were mild in most patients. Severe colitis, a drug-associated AE observed in ipilimumab-treated patients (Beck et al., 2006), was poorly demonstrated with anti-PD-L1.

  As noted above for anti-PD-1 immunotherapy, another important property of anti-PD-L1 treatment is the persistence of response across multiple tumor types. This is particularly striking in view of the patient's advanced disease and previous treatment in the current study. Although not directly compared, this persistence appears to be better than that observed with most chemotherapies and kinase inhibitors used to treat these cancers.

  Since peripheral blood T-cells express PD-L1, in vivo RO with BMS-963559 can be evaluated as a pharmacokinetic criterion. The median RO was 65.8%, 66.2%, and 72.4% for the dose tested. Although these studies provide direct assessment and evidence of target involvement in patients treated with BMS-936559, the relationship between RO and peripheral tumor microenvironment in peripheral blood remains poorly understood.

  Based on the clinical data described herein, the present application provides a method for immunotherapy of a subject with cancer, comprising a therapeutically effective amount of an anti-PD-L1 Ab of the invention or There is provided a method comprising administering to a subject a composition comprising the antigen binding portion thereof. The application also provides a method of inhibiting tumor cell growth in a subject, comprising administering to the subject an anti-PD-L1 Ab or an antigen-binding portion thereof of the invention. In a preferred embodiment, the subject is a human. In one embodiment, the Ab or antigen binding portion thereof is of the IgG1 or IgG4 isotype. In other embodiments, the Ab or antigen binding portion thereof is a mAb or antigen binding portion thereof. In a further aspect, the Ab or antigen binding portion thereof is a chimeric, humanized or human Ab or antigen binding portion thereof. In a preferred embodiment for treating a human patient, the Ab or antigen binding portion thereof is a human Ab or antigen binding portion thereof.

The clinical trials described in the examples used anti-PD-L1 HuMAb BMS-936559 to treat cancer. While BMS-936559 (referred to as HuMAb 12A4 in U.S. Patent No. 7,943,743) was selected as the lead anti-PD-L1 Ab for participation in clinical trials, but notably, Anti-PD-L1 Ab of the present invention specifically binds with high affinity to 12A4 functional properties that are important for the therapeutic activity of 12A4, such as T-cell proliferation in MLR assays, Increasing IL-2 secretion and interferon-γ production, inhibiting PD-L1 binding to PD-1, and reversing the inhibitory effect of T regulatory cells on T cell effector cells and / or dendritic cells It is to share what you do. Furthermore, certain anti -PD-L1 Ab of the present invention, i.e. 1B12,7H1 and 12B7 comprise _{V H} and V _kappa region having a sequence derived from the _V H 1-69 and V _kappa L6 germline sequence, respectively It is structurally related to 12A4. In addition, at least 12B7, 3G10, 1B12 and 13G4 cross-compete with 12A4 for binding to the same epitope region of hPD-L1, whereas 5F8 and 10A5 bind to the same or overlapping 12A4 epitope region as 12A4. (Examples 2 and 3). Thus, the preclinical characterization of 12A4 and other anti-PD-L1 HuMabs suggests that the methods of treating cancer provided herein use any of the broad genera of anti-PD-L1 Abs of the present invention. It is shown that it can be implemented.

This application is therefore, (a) a heavy chain variable region comprising the consecutively linked amino acids having a sequence derived from a human V _H 1-18 germline sequence, and sequences from human V _kappa L6 germline sequence A light chain variable region comprising contiguously linked amino acids having a sequence derived from a human V _H 1-69 germline sequence; and A light chain variable region comprising a continuously linked amino acid having a sequence derived from a _Vκ L6 germline sequence; (c) a continuously linked sequence having a sequence derived from a human V _H 1-3 germline sequence a light chain variable region comprising the consecutively linked amino acids having the sequence from the heavy chain variable region, and a human V _kappa L15 germline sequence comprises the amino acid; (d) human V _H 1-69 germline distribution A light chain variable region comprises a heavy chain variable region and consecutively linked amino acids having a sequence derived from a human V _kappa A27 germline sequence comprises consecutively linked amino acids having a sequence derived from; (e) heavy chain variable region and consecutively linked amino acids having a sequence derived from a human V _kappa L15 germline sequence comprises consecutively linked amino acids having a sequence derived from a human V _H 3-9 germline sequence origin or (f) a heavy chain variable region comprising the consecutively linked amino acids having a sequence derived from a human V _H 3-9 germline sequence, and the human V _kappa L18 germline sequence; a light chain variable region comprising the An immunotherapeutic method comprising administering to a patient an anti-PD-L1 Ab comprising a light chain variable region comprising a continuously linked amino acid having the sequence .

  In one embodiment, the anti-PD-L1 Ab or antigen-binding portion thereof administered to the patient has a (a) contiguous sequence having the sequence set forth in SEQ ID NO: 15 for binding to PD-L1. A human heavy chain variable region comprising an amino acid linked to SEQ ID NO: 25 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 25; (b) set forth in SEQ ID NO: 16 A human heavy chain variable region comprising a contiguously linked amino acid having the sequence set forth and a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 26; (c) A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 17 and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 27 (D) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 18 and a sequence set forth in SEQ ID NO: 28 A human light chain variable region comprising contiguously linked amino acids; (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 19 and SEQ ID NO: 29. A human light chain variable region comprising a contiguously linked amino acid having the sequence set forth; (f) a human heavy chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 20 A human light chain variable region comprising a region and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 30; (g) a continuously linked sequence having the sequence set forth in SEQ ID NO: 21 A human heavy chain variable region comprising a mutated amino acid and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 31; (h) set forth in SEQ ID NO: 22 A human heavy chain variable region comprising a contiguously linked amino acid having the sequence and a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 32; (i) SEQ ID NO: : A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 23 and a human light chain variable comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 33 Or (j) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 24 and the sequence set forth in SEQ ID NO: 34. Continuously linked referenced Ab or reference antigen-binding portion thereof, comprising a human light chain variable region comprising the amino acid to cross-compete. In a preferred embodiment, the Ab or antigen-binding portion thereof comprises a human heavy chain variable region and sequence comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 16 for binding to PD-1. Cross-competes with a reference Ab or a reference antigen-binding portion thereof comprising a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 26.

  In one preferred embodiment of the immunotherapy methods described herein, the anti-PD-L1 Ab or antigen-binding portion thereof administered to the subject comprises (a) a sequence set forth in SEQ ID NO: 15. A human heavy chain variable region comprising contiguously linked amino acids and a human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 25; (b) SEQ ID NO: 16 A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 26 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 26; (C) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 17 and a contiguous sequence having the sequence set forth in SEQ ID NO: 27 A human light chain variable region comprising a linked amino acid; (d) a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 18 and a human light chain variable region comprising the sequence set forth in SEQ ID NO: 28. A human light chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 19; and (e) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 19. A human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 29; (f) including a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 20 A human heavy chain variable region comprising a continuously linked amino acid having a human heavy chain variable region and a sequence set forth in SEQ ID NO: 30; (g) a sequence set forth in SEQ ID NO: 21; A human heavy chain variable region comprising a contiguously linked amino acid having a human light chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 31; (h) SEQ ID NO: 22 A human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 32 and a human light chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 32; (I) combining a human heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 23 and a continuously linked amino acid having the sequence set forth in SEQ ID NO: 33 (J) a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 24; Including human light chain variable region comprising the consecutively linked amino acids having the sequence set. In a more preferred embodiment, the anti-PD-L1 Ab or antigen-binding portion is a human heavy chain variable region comprising contiguously linked amino acids having the sequence set forth in SEQ ID NO: 16 and set forth in SEQ ID NO: 26. And a human light chain variable region comprising contiguously linked amino acids having the sequence of interest.

The wide spectrum of cancers treatable by anti-PD-L1 immunotherapy Because NSCLC is believed to have a poor response to immune-based treatment, the clinical activity of anti-PD-L1 in patients with advanced NSCLC As well as the activity of anti-PD-1 in patients with this disease, it was surprising and unexpected (Holt and Disis, 2008; Holt et al., 2011). The present clinical data obtained with BMS-936559, an anti-PD-L1 Ab of the present invention, indicate that immunotherapy based on PD-1 blockade could not only be applied to "immunogenic" tumor types, such as MEL and RCC. Demonstrate and extend the evidence obtained using anti-PD-1 Ab that is also effective in a wide range of cancers, including treatment-refractory metastatic NSCLC that is not generally considered to respond. Preferred cancers that can be treated using the anti-PD-L1 Abs of the invention include MEL (eg, metastatic malignant melanoma), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer (OV), Includes gastric (GC), breast (BC), pancreatic (PC) and esophageal carcinomas. Further, the invention includes refractory or recurrent malignancies whose growth can be inhibited using the anti-PD-L1 Ab of the invention.

  Thus, examples of cancers that can be treated using anti-PD-L1 Abs in the methods of the present invention based on the indications provided by the very broad applicability of anti-PD-L1 immunotherapy provided herein. For bone, skin, head and neck, breast, lung, skin or intraocular melanoma, kidney, uterine, castration-resistant prostate, colon, transrectal, anal area, gastric cancer , Testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, ovarian carcinoma, gastrointestinal tract and breast, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the esophagus Cancer of the small intestine, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia including acute myeloid leukemia, chronic myeloid leukemia , Acute lymphoblastic leukemia, chronic lymphocytic leukemia, pediatric Shape tumor, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, tumor of the spinal axis, brain stem Glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, multiple myeloma, environment-induced cancer including asbestos-induced cancer, metastatic cancer, and any of the above cancers Including combinations of The invention is also applicable to the treatment of metastatic cancer.

Combination Therapy with Anti-PD-L1 Ab Optionally, the Ab against PD-L1 may be an immunogen, eg, a preparation of cancerous cells, a purified tumor antigen (including recombinant proteins, peptides, and carbohydrate molecules). , Antigen-presenting cells, such as dendritic cells with tumor-associated antigens, and cells transfected with a gene encoding an immunostimulatory cytokine (He et al., 2004). Non-limiting examples of tumor vaccines that can be used include those that express a peptide of a melanoma antigen, such as a peptide of gp100, a MAGE antigen, Trp-2, MART1 and / or tyrosinase, or the cytokine GM-CSF. Includes transfected tumor cells. PD-L1 blockade is also indicated by chemotherapy regimens, radiation therapy, surgical procedures, standard cancer treatments including hormonal loss and angiogenesis inhibitors, and other immunotherapeutic Abs (eg, anti-PD-1, anti-CTLA-4 or It can be effectively combined with anti-LAG-3 Ab).

Use of anti-PD-L1 Abs This application describes the manufacture of a medicament for inhibiting signaling from the PD-1 / PD-L1 pathway, thereby enhancing the endogenous immune response in a subject suffering from cancer. There is provided the use of any of the anti-PD-L1 Abs or antigen-binding portions thereof of the invention for the treatment of The present application also provides for any of the anti- antibodies of the present invention for the manufacture of a medicament for immunotherapy of a subject suffering from cancer, including disrupting the interaction between PD-1 and PD-L1. Provided is the use of a PD-L1 Ab or an antigen-binding portion thereof. The present application relates to the medical application of any anti-PD-L1 Ab or antigen-binding portion thereof of the invention corresponding to all aspects of the method of treatment using an anti-PD-L1 Ab described herein. Provide use.

  The present application also provides for a subject suffering from cancer, including enhancing an endogenous immune response in a subject suffering from cancer by inhibiting signaling from the PD-1 / PD-L1 pathway. Provided is an anti-PD-L1 Ab, or antigen-binding portion thereof, of the invention for use in treating. The present application provides an anti-PD-L1 Ab or antigen thereof of the invention for use in immunotherapy of a subject suffering from cancer, comprising disrupting the interaction between PD-1 and PD-L1. A binding moiety is further provided. These Abs can be used in enhancing the endogenous immune response to all of the cancers described herein, or in immunotherapy of all of the cancers described herein. . In a preferred embodiment, the cancer is MEL (eg, metastatic malignant MEL), RCC, squamous NSCLC, non-squamous NSCLC, CRC, ovarian cancer (OV), gastric cancer (GC), breast cancer (BC), pancreatic carcinoma (PC ) And esophageal carcinoma.

Validation of cancer immunotherapy by immune checkpoint blockade Significant impact of clinical activity of immune checkpoint blockade results in significant endogenous immune responses to tumor antigens that mediate clinical tumor regression upon checkpoint inhibition It can be used therapeutically. Indeed, there is evidence for a mechanism called adaptive resistance in which inhibitory ligands, such as PD-L1, are induced in response to an immune challenge (Gajewski et al., 2010; Taube et al., 2012). This possible mechanism of immune tolerance by tumors suggests that PD-1 / PD-L1-directed therapy may provide a synergistic effect with other treatments that increase endogenous anti-tumor immunity. Follow-up studies have demonstrated that patients continue to show tumor control after cessation of PD-1 / PD-L1 pathway blockade (Lipson et al., 2013). Such tumor controls can be reflected in the production of effective immune memory that allows for a sustained anti-tumor immune response and sustained control of tumor growth.

  The data described herein in clinical trials of Abs that block the immunomodulatory receptor PD-1 and one of its cognate ligands, PD-L1, are unprecedented. These data demonstrate the greatest clinical experience to date with PD-1 pathway-directed cancer immunotherapy, and the safety, tolerability, and initial clinical activity of anti-PD-L1-directed agents Make up the first report to be made. These findings indicate that both anti-PD-1 and anti-PD-L1 have favorable overall safety profiles and NSCLC, a tumor that has not been historically considered to respond to immunotherapy, And provide clear evidence of clinical activity across a variety of cancers, including tumors known to respond to immunotherapy including MEL, RCC and OV. Thus, these data strongly demonstrate the PD-1 / PD-L1 pathway as a key target for therapeutic intervention in cancer.

  The striking similarities observed between the patterns of clinical activity observed with anti-PD-1 and anti-PD-L1 mAbs in the tumor types analyzed to date indicate that tumor immunoresistance and therapeutic intervention Demonstrate the general importance of the PD-1 / PD-L1 signaling pathway as a target for The molecular interactions blocked by these two Abs are not identical, but regardless of the details of the mechanism, both anti-PD-1 and the anti-PD-L1 Ab of the present invention can be used in a wide variety of cancers. It has been clearly demonstrated herein to be useful in treating afflicted patients.

Infections Other methods of the invention are used to treat patients that have been exposed to a particular toxin or pathogen. For example, another aspect of the present application involves administering to a subject an anti-PD1 or anti-PD-L1 Ab of the invention, or an antigen-binding portion thereof, such that the subject is treated for an infectious disease. And methods of treating an infectious disease in a subject. Preferably, the Ab is a human anti-human PD-1 or PD-L1 Ab (eg, any human Ab described herein). Alternatively, the Ab is a chimeric or humanized Ab.

  Similar to tumor applications as described above, Ab-mediated PD-1 or PD-L1 blockade may be used alone or in combination with vaccines as an adjuvant to enhance the immune response to pathogens, toxins, and / or self antigens. , Can be used. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which there is currently no effective vaccine, or those for which conventional vaccines are not completely effective. These include, but are not limited to, HIV, hepatitis (A, B, and C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. PD-1 and / or PD-L1 blockade is particularly useful for infections established by agents that exhibit altered antigen during infection, such as HIV. Novel epitopes on these antigens are recognized as foreign upon administration of anti-human PD-1 or PD-L1, and therefore exert a strong T cell response that is not suppressed by negative signals through the PD-1 / PD-L1 pathway. Induce.

  In the above method, PD-1 or PD-L1 blockade can be combined with other forms of immunotherapy, eg, cytokine treatment (eg, administration of interferon, GM-CSF, G-CSF or IL-2).

Kits Pharmaceutical kits comprising a combination of an anti-PD-1 and / or anti-PD-L1 Ab, or anti-PD-1 and anti-CTLA-4 Ab of the invention for therapeutic use, and immunotherapy. A diagnostic kit comprising an anti-PD-L1 Ab of the present invention for assaying PD-L1 expression on membranes, or as a biomarker for screening patients to predict the efficacy of an immunotherapeutic agent. Included kits are also within the scope of the present invention. Kits generally include a label that supports the intended use of the contents of the kit and instructions for use. The term label includes any written material, or described material provided on or with the kit, or others attached to the kit. In one embodiment of the pharmaceutical kit, the anti-PD1 and / or anti-PD-L1 Ab may be packaged with the other therapeutic agent in a unit dosage form. In one embodiment of the diagnostic kit, the anti-PD-L1 Ab may be packaged with other reagents for performing an assay to detect and / or quantify PD-L1 expression.

In one preferred embodiment, the pharmaceutical kit comprises nivolumab, which is an anti-human PD-1 HuMAb. In another preferred embodiment, the pharmaceutical kit comprises BMS-936559, an anti-human PD-L1 HuMAb. In yet another preferred embodiment, the pharmaceutical kit comprises ipilimumab, which is an anti-human CTLA-4 HuMAb. In one preferred embodiment, the diagnostic kit rabbit anti including _{V H} and V _kappa region amino acid sequences are described in SEQ ID NO: 35 and 36 - human PD-L1 mAb, including 28-8. In another preferred embodiment, the diagnostic kit comprises the mouse anti-human PD-L1 mAb, 5H1 (Dong et al., 2002).

PD-L1 Biomarkers to Predict Anti-PD-1 Efficacy A particular challenge in cancer immunotherapy is the identification of predictive biomarkers based on mechanisms that allow guidance on patient selection and treatment management. The data described in the Examples below show that cell surface PD-L1 expression in tumors predicts the efficacy of immunotherapy with anti-PD-1 and potential other immune checkpoint inhibitors. Or a useful molecular marker for selecting patients for the immunotherapy.

  There are conflicting reports in the literature on the clinical significance of PD-L1 expressed in tumors. Several studies have concluded that PD-L1 expression in tumors correlates with poor prognosis for patients. See, for example, Hino et al. (2010) (MEL); Hamanishi et al. (2007) (OV); Thompson et al. (2006) (RCC). These findings indicate that the interaction of PD-L1 on tumor cells and PD-1 on T cells helps to abolish the immune response to tumors and causes immune escape from tumor-specific T cells. And can be explained rationally (Blank et al., 2005). However, in contrast to the above study, Gadiot et al. (2011) and Taube et al. (2012) have recently reported that PD-L1 expression in melanoma tumors correlates with a better propensity for survival. These seemingly conflicting data indicate that relatively few patients were analyzed, different histological subtypes tested, or different methodologies used, eg, the use of different Abs to stain PD-L1. , The use of frozen versus paraffin-embedded material for IHC, and the detection of PD-L1 membrane and / or cytoplasmic staining. Taube et al. (2012) describe that PD-L1 is a type I transmembrane molecule, and the cytoplasmic presence of PD-L1 indicates that intracellular storage of this polypeptide can be located on the cell surface upon appropriate stimulation. Has been hypothesized to be biologically relevant cell surface PD-L1 expression as a potential biomarker for predicting clinical response to PD-1 blockade. See also Brahmer et al. (2010), describing preliminary evidence of a correlation between PD-L1 expression of membranes and anti-PD-1 efficacy, obtained in a small sample size of only 9 patients. The data described in the Examples below on the use of membrane PD-L1 expression as a biomarker for anti-PD-1 efficacy, obtained from analysis of a large sample, shows that PD-L1 expression Hypothesis could be used as a biomarker to predict anti-PD-1 clinical response and to screen patients to identify suitable candidates for immunotherapy with anti-PD-1 Ab Prove that. Although the utility of this biomarker to screen patients for anti-PD-1 immunotherapy or predict clinical response to anti-PD-1 immunotherapy has been demonstrated, PD-L1 expression is It can also potentially be applied more broadly as a combination biomarker for other types of inhibitors of inhibitory immune regulators.

  Specifically, membrane PD-L1 expression was assayed using an automated IHC protocol and a rabbit anti-hPD-L1 Ab. Significantly, in the first set of data analyzed (see Example 8), patients with cell surface PD-L1-negative tumors (MEL, NSCLC, CRC, RCC and CRPC) are anti-PD-1 Abs. No OR was experienced after treatment with nivolumab. In contrast, cell surface expression of PD-L1 on tumor cells in pretreatment biopsies may be associated with an increased rate of OR in patients treated with nivolumab. Endogenous antitumor that is part of the host inflammatory response, although tumor cell expression of PD-L1 can be driven by a constitutive oncogenic pathway but can be maintained unless released by blockade of the PD-1 / PD-L1 pathway It may be reflected in "adaptive immune tolerance" in response to an immune response (Taube et al., 2012). This new concept of adaptive immune resistance in cancer immunology suggests that inhibitory ligands, such as PD-L1, are induced in response to an immune challenge (Gajewski et al., 2010; Taube et al., 2012). The significant effect of the clinical activity of immune checkpoint blockade described herein is that a significant endogenous immune response to tumor antigens occurs, and these responses mediate clinical tumor regression upon checkpoint inhibition. It can be used therapeutically. This possible mechanism of immune tolerance by tumors suggests that PD-1 / PD-L1-directed therapy may provide a synergistic effect with other treatments that increase endogenous anti-tumor immunity.

Assay for Cell Surface PD-L1 Expression by Automated IHC As described in the Examples, an automated IHC method was developed to assay PD-L1 expression on the surface of cells in FFPE tissue specimens. The present application is a method for detecting the presence of human PD-L1 antigen in a test tissue sample, or quantifying the level of human PD-L1 antigen or the percentage of cells in a sample that expresses the antigen, the method comprising the steps of: A method comprising contacting a test sample and a negative control sample with a mAb that specifically binds human PD-L1 under conditions that allow for the formation of a complex between the moiety and human PD-L1. . Preferably, the test and control tissue samples are FFPE samples. Next, complex formation is detected, and a difference in complex formation between the test sample and the negative control sample indicates the presence of the human PD-L1 antigen in the sample. Various methods are used to quantify PD-L1 expression.

  In certain embodiments, the automated IHC method comprises: (a) deparaffinizing and rehydrating the mounted tissue section in an autostainer; (b) antigen retrieval heated to 110 ° C. for 10 minutes. Retrieving the antigen using a decloaking chamber and pH 6 buffer; (c) setting the reagents on an autostainer; and (d) neutralizing endogenous peroxidase in the tissue specimen Moving the autostainer to include steps; blocking non-specific protein binding sites on the slide; incubating the slide with primary Ab; incubating with a post-primary blocker; Incubating with NovoLink Polymer; adding chromogen substrate and developing color; and pairing with hematoxylin Including staining.

To assess PD-L1 expression in tumor tissue samples, a pathologist observes the number of membrane PD-L1 ⁺ tumor cells in each region under a microscope and determines the percentage of cells that are positive in mind ( mentally) and then average them to get the final percentage. Various staining intensities are defined as 0 / negative, 1 + / weak, 2 + / medium, and 3 + / strong. Generally, percentage values are initially assigned to 0 and 3+ buckets, and then the intermediate 1+ and 2+ intensities are considered. In highly heterogeneous tissues, specimens are classified into zones, each zone is scored separately, and then combined into a single set of percentage values. The percentage of negative and positive cells for different staining intensities is determined from each area and the median is given to each zone. Final percentage values are given to the tissue for each staining intensity category: negative, 1+, 2+, and 3+. The sum of all staining intensities must be 100%.

  Staining is also assessed in tumor infiltrating inflammatory cells, such as macrophages and lymphocytes. Macrophages serve as internal positive controls, since in most cases staining is observed in most macrophages. It is not necessary to stain at 3+ intensity, but the absence of macrophage staining should be considered to rule out any technical failure. Macrophages and lymphocytes are assessed for cell membrane staining and are only scored for all samples as positive or negative for each cell category. The staining is also characterized according to the external / internal tumor immune cell designation. "Internal" means that the immune cells are within the tumor tissue and / or on the boundaries of the tumor area without being physically inserted into the tumor cells. "External" means no physical association with the tumor, and the immune cells are found in the periphery associated with the connective or some related adjacent tissue.

  In one aspect of these scoring methods, the sample is scored by two independently operating pathologists, and the scores are then integrated. In one other embodiment, the identification of positive and negative cells is scored using appropriate software.

Histo-score is used as a further quantitative measure of IHC data. Histo-score is calculated as follows:
Histo score = [(% tumor x1 (low intensity)) + (% tumor x2 (medium intensity)) + (% tumor x3 (high intensity)]

  To determine the histoscore, the pathologist estimates the percentage of stained cells in each intensity category in the specimen. Because the expression of many biomarkers is heterogeneous, the histoscore is a more accurate indication of total expression. The final histo-score range is 0 (no expression) to 300 (maximum expression).

  An alternative means of quantifying PD-L1 expression in a test tissue sample IHC is to determine an adjusted inflammatory score (AIS) defined as the density of inflammation multiplied by the percent PD-L1 expression by tumor-infiltrating inflammatory cells (Taube et al., 2012).

Cancer Immunotherapy with Anti-PD-1 Including a Patient Selection Step The present application also provides (a) (i) optionally providing a test tissue sample obtained from a patient having a tissue cancer, wherein the test tissue sample comprises The test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (ii) assessing the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample; and (iii) Immunotherapy, including selecting a subject as a suitable candidate based on an assessment that the percentage of cells expressing PD-L1 on the cell surface exceeds a predetermined threshold level, eg, anti-PD-1 Ab Selecting a subject that is a suitable candidate for the administration of a drug; and (b) a therapeutically effective amount of an agent that inhibits signaling from an inhibitory immune regulator, eg, anti-PD- A composition comprising ab, comprising administering to a subject that has been selected, provides a method for immunotherapy of a subject suffering from cancer.

  There is evidence that membrane PD-L1 expression is an alternative to the endogenous anti-tumor immune response that is part of the host inflammatory response (Gajewski et al., 2010; Taube et al., 2012). Therefore, cell surface expression of PD-L1 in tumor and / or inflammatory cells in the intratumoral microenvironment is not only a treatment with anti-PD-1 Ab, but also with treatment with anti-PD-L1 Ab and PD It can be a marker for selecting cancer patients that would also benefit from treatments targeting inhibitory immune regulatory pathways other than the -1 / PD-L1 pathway. For example, cell surface expression of PD-L1 in tumors and / or tumor-infiltrating inflammatory cells can be determined by immune checkpoints, such as PD-L1, cytotoxic T-lymphocyte antigen-4 (CTLA-4), B and T lymphocytes. Sphere attenuator (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), killer immunoglobulin-like receptor (KIR), killer cell lectin-like Appropriate agents that target, destroy or inhibit signaling from receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), and CD160, eg, would benefit from immunotherapy with Abs It can be used as a marker to identify or select cancer patients (Pardoll, 2012; Baitsch et al., 2012). In one preferred embodiment, the inhibitory immune regulator is a component of the PD-1 / PD-L1 signaling pathway. In another preferred embodiment, the inhibitory immunoregulator is an anti-PD-1 Ab of the invention. In yet another preferred embodiment, the inhibitory immunoregulator is an anti-PD-L1 Ab of the invention.

  Any method of immunotherapy that involves assaying PD-L1 expression, ie, using a PD-L1 expression biomarker, involves selecting patients who are or are not suitable for anti-PD-1 immunotherapy. When described below, or when including the administration of an anti-PD-1 Ab for immunotherapeutic purposes, these methods may be performed using an inhibitory immune regulator (eg, CTLA-4, BTLA, TIM3, LAG3). Or KIR), or immunotherapy with the administration of inhibitors of its components or ligands, or should be understood to be more widely used in the selection of patients suitable or inappropriate for such administration. Further, in any method that includes measuring PD-L1 expression in a test tissue sample, it should be understood that the step of providing a test tissue sample obtained from the patient is an optional step. That is, in one embodiment, the method includes this step, and in another embodiment, the step is not included in the method. In one preferred embodiment, the step of "assessing" to identify or determine the number or percentage of cells expressing PD-L1 on the cell surface in a test tissue sample comprises the step of assaying for PD-L1 expression It should be understood that the transformation is performed by a method, for example, by performing a reverse transcriptase-polymerase chain reaction (RT-PCR) assay or an IHC assay. In one other embodiment, without the transformation step, PD-L1 expression is assessed, for example, by reviewing a report of test results from a laboratory. In one embodiment, the method of assessing PD-L1 expression or a method comprising assessing PD-L1 expression comprises selecting suitable candidates for immunotherapy and / or administering the immunotherapeutic agent to the patient. Provides an intermediate result that can be provided to a physician or other physician for use in administering to the physician. In one aspect, the step of providing an intermediate result may be performed by a physician or a person acting under the guidance of a physician. In other embodiments, these steps are performed by an independent laboratory or by an independent person, for example, an experimental assistant.

  The application also provides (a) (i) optionally, a test tissue sample obtained from a patient having a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (Ii) assessing the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample; and (iii) determining the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample in advance. Selecting an inhibitor based on an assessment of being below the determined threshold level, such as an inhibitor of an inhibitory immune regulator, e.g., an anti-PD-1 Ab, as inappropriate for immunotherapy. Selecting a subject that is not suitable for treatment with an inhibitory agent, eg, anti-PD-1 Ab immunotherapy; and (b) an inhibitory immune regulator A method of treating a subject suffering from cancer, comprising administering to a selected subject a standard therapeutic agent other than an inhibitor of the present invention, for example, an anti-PD-1 Ab.

Measuring PD-L1 Expression In one embodiment of any of the methods of the present application, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 RNA. In a further aspect, the presence of PD-L1 RNA is determined by RT-PCR, in situ hybridization or RNase protection. In other embodiments, the percentage of cells expressing PD-L1 is assessed by performing an assay to determine the presence of PD-L1 polypeptide. In a further aspect, the presence of the PD-L1 polypeptide is determined by immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), in vivo imaging, or flow cytometry. In a preferred embodiment, PD-L1 expression is assayed by IHC. Flow cytometry may be particularly suitable for assaying PD-L1 expression in cells of a hematological tumor. In a preferred embodiment of all these methods, cell surface expression of PD-L1 is assayed using, for example, IHC or in vivo imaging.

  Imaging technology has provided an important tool in cancer research and treatment. Molecular imaging systems such as positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence reflectance imaging (FRI), fluorescence-mediated tomography (FMT), bioluminescence Recent developments in imaging (BLI), laser scanning confocal microscopy (LSCM) and multiphoton microscopy (MPM) will pioneer the use of these techniques in cancer research. Some of these molecular imaging systems allow clinicians not only to see if a tumor is in the body, but also to determine the specific molecules, cells, and biological properties that affect tumor kinetics and / or responsiveness to therapeutic agents. It also allows to visualize the expression and activity of the process (Condeelis and Weissleder, 2010). Ab specificity, in addition to PET sensitivity and resolution, makes immuno-PET imaging particularly attractive for monitoring and assaying antigen expression in tissue samples (McCabe and Wu, 2010; Olafsen et al., 2010). In one embodiment of any of the methods of the present application, PD-L1 expression is assayed by immuno PET imaging.

  In one embodiment of any of the methods of the present application, the percentage of cells in a test tissue sample expressing PD-L1 is determined by an assay to determine the presence of PD-L1 polypeptide on the surface of cells in the test tissue sample. It is evaluated by performing. In one embodiment, the test tissue sample is a FFPE tissue sample. In one preferred embodiment, the presence of a PD-L1 polypeptide is determined by an IHC assay. In a further aspect, the IHC assay is performed using an automated process. In a further aspect, the IHC assay is performed using an anti-PD-L1 mAb that binds to a PD-L1 polypeptide.

Abs that specifically bind to PD-L1 expressed on the cell surface in FFPE tissues
Abs can bind antigen in fresh tissue, but fail to fully recognize the antigen in FFPE tissue samples. This phenomenon, well known in the art, is believed to be primarily due to intramolecular and intermolecular cross-linking of the polypeptide induced by formalin fixation that alters the epitope recognized by the Ab (Sompuram et al., 2006). ). In addition, several factors known to affect staining in FFPE tissue, such as variable time to fixation, insufficient fixation period, differences in fixative used, tissue processing, Ab clone and dilution, Antigen retrieval, detection systems, and interpretation of results using different threshold points are important variables that can affect tissue antigenicity and IHC measurements (Bordeaux et al., 2010). In particular, the lack of an anti-human PD-L1 Ab that stains PD-L1 in FFPE specimens has been noticed in the art (Hamanishi et al., 2007). Taube et al. (2012) and Gadiot et al. (2011) also report difficulties in identifying anti-PD-L1 Abs that specifically bind to PD-L1 in FFPE tissues, and conflicting test results for the same Abs. doing. Our own analysis of five commercially available anti-hPD-L1 Abs failed to distinguish these FFPE cells that expressed PD-L1 from cells that did not express PD-L1 (See Example 9, Table 7). Thus, conflicting results reported by different groups in the association of PD-L1 expression on tumor prognosis are due, in part, to the anti-PD-L1 used to detect PD-L1 polypeptide in FFPE tissue samples. It may reflect the unique ability of Ab. Therefore, to detect hPD-L1 on the surface of cells using the IHC assay in FFPE tissues, an anti-hPD-L1 Ab that specifically binds to PD-L1 expressed on the cell surface in FFPE tissue samples There is a need for

The present application provides a mAb or antigen-binding portion thereof that specifically binds to a cell surface expressed PD-L1 antigen in an FFPE tissue sample. In a preferred embodiment, the mAb or antigen-binding portion thereof does not bind to cytoplasmic PD-L1 polypeptide or exhibits a very low level of background binding in FFPE tissue samples. In one other embodiment, the presence or absence of specific binding to cell surface expressed or cytoplasmic PD-L1 polypeptide is detected by immunohistochemical staining. In one preferred aspect of the invention, the mAb or antigen binding portion is a rabbit Ab or a portion thereof. In other preferred embodiments, the mAb is a rabbit mAb designated as 28-8, 28-1, 28-12, 29-8 or 20-12. In a more preferred embodiment, the mAb is a rabbit mAb designated 28-8 or an antigen binding portion thereof. In a further aspect, the mAb is a heavy chain variable region (V _H ) comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 35 and a contiguous sequence having the sequence set forth in SEQ ID NO: 36. An Ab comprising a light chain variable region (V _κ ) comprising amino acids linked together. In other embodiments, mAb is SEQ ID NO: 35 CDRl, CDR2 and CDR3 domains in _{V H} having the sequence set forth in and SEQ ID NO: 36 CDRl of V _kappa having the sequence set forth in, CDR2 and Includes CDR3 domain.

  It is known in the art that rabbit Abs have certain advantages over mouse Abs. For example, rabbit Abs generally show a greater variety of epitope recognition, improved immune response to small size epitopes, and higher specificity and affinity compared to mouse Abs (see, eg, Fischer et al.). Cheang et al., 2006; Rossi et al., 2005). For example, the lower immunodominance of rabbits and the larger B cell repertoire cause better epitope recognition as compared to mouse Abs. In addition, the high specificity of rabbit antibodies and novel epitope recognition leads to successful recognition of post-translational modifications (Epitomics, 2013). In addition, a number of protein targets associated with signal transduction and disease are highly conserved among mice, rats and humans, and thus can be recognized as autoantigens by the mouse or rat host, which translates them into lower immunogens. Make it sex. This problem is circumvented by producing Ab in rabbits. Furthermore, in applications where two antigen-specific Abs are required, it is more convenient to derive Abs from two different species. Thus, for example, rabbit Abs, eg, other Abs that can mark immune cells (eg, macrophages and lymphocytes) that also express PD-L1 at 28-8 (because the best immunomarking Ab is the mouse Ab) , May be mouse Ab). Thus, rabbit anti-hPD-L1 mAbs, eg, 28-8, are particularly suitable for IHC assays to detect PD-L1 expressed on the surface in FFPE tissue samples, and exceed mouse Abs, eg, 5H1 Potentially has obvious benefits.

  As described in Example 9, a large number (185) of Ab multiclones from both rabbit and mouse immunizations were screened, and no mouse Abs, but only 10 rabbit Ab multiclones were hPD- The membrane morphology of L1 was specifically detected. After more extensive screening by multiple rounds of IHC, 15 purified rabbit subclones were selected based on the specificity and intensity of their staining (see Table 5). After screening by IHC in FFPE tissues and further characterization of the antibodies to determine binding affinity and cross-competition by surface plasmon resonance, mAb28-8 binds to membrane PDF-L1 with high affinity and specificity and The Ab with the best combination of low background staining was selected.

  In one aspect of the invention, the mAb or antigen-binding portion cross-competes with mouse mAb 5H1 for binding to PD-L1, which allows these antibodies to bind to the same epitope region of PD-L1. Indicates that In one other aspect, the mAbs or antigen-binding portions thereof do not cross-compete with mouse mAb 5H1 for binding to PD-L1, indicating that they do not bind to the same epitope region of PD-L1. .

  The application also provides a nucleic acid encoding any of the rabbit anti-hPD-L1 Abs or portions thereof described herein.

Immunotherapeutic Methods Involving Measurement of Cell Surface PD-L1 Expression The availability of rabbit Abs that specifically bind membrane PD-L1 with high affinity in FFPE tissue specimens has been demonstrated for PD on cell surfaces in FFPE tissue samples. -Facilitating a method comprising the step of detecting an L1 polypeptide. Accordingly, the present application also provides (a) (i) optionally a test tissue sample obtained from a patient having a cancer of FFPE tissue, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells (Ii) of cells expressing PD-L1 on the cell surface in a test tissue sample by IHC using a rabbit anti-human PD-L1 Ab, eg, mAb 28-8, which binds to PD-L1. Assessing the proportion; and (iii) selecting the subject as a suitable candidate based on the assessment that the proportion of cells expressing PD-L1 on the cell surface in the test tissue sample exceeds a predetermined threshold level Selecting a subject that is a suitable candidate for immunotherapy comprising: and (b) inhibiting a therapeutically effective amount of an inhibitory immune regulator Agents, for example, comprises administering to a subject that is selected a composition comprising an anti -PD-1 Ab, a method for immunotherapy of a subject suffering from cancer.

  In one embodiment of the method of using IHC to assay PD-L1 expression in FFPE tissue, an automated IHC assay is used. The automated IHC process is performed in an autostainer, (a) deparaffinizing the FFPE sample with xylene, and rehydrating the sample; (b) activating the antigen using an antigen stimulator (C) blocking non-specific protein binding sites by incubation with Protein Block; (d) incubating the sample with a primary anti-PD-L1 Ab; (e) polymeric horseradish peroxidase (HRP). (F) detecting bound secondary Ab, including staining with 3,3'-diaminobenzidine (DAB) chromogen; and / or (g) hematoxylin And counterstaining. This automated IHC process has been optimized by minimizing the number of steps, optimizing the incubation time, and selecting primary Abs, blocking and detection reagents that produce strong specific staining with low levels of background staining. In a preferred embodiment of this automated IHC assay, the primary anti-PD-L1 Ab is rabbit mAb 28-8 or mouse mAb 5H1. In one aspect of the invention, this IHC assay, and any other IHC assays described herein for measuring PD-L1 expression, can be used as part of an immunotherapy method. In other embodiments, any of the IHC methods described herein are independent of any therapeutic process that requires administration of a therapeutic agent, ie, as a diagnostic method for assaying PD-L1 expression. Used alone.

  In one aspect of any of the immunotherapy methods described herein, the Ab administered to the selected subject is any of the anti-PD-1 or anti-PD-L1 Abs of the invention or antigens thereof. It is a connecting part. In one preferred embodiment, the subject is a human. In another preferred embodiment, the Ab is a human Ab or an antigen binding portion thereof. In a more preferred embodiment, the anti-PD-1 Ab is nivolumab and the anti-PD-L1 Ab is BMS-936559. In one other embodiment, the anti-PD-1 Ab is an Ab or an antigen-binding portion thereof that cross-competes with nivolumab for binding to PD-1, and the anti-PD-L1 Ab binds to PD-L1. An Ab or antigen-binding portion thereof that cross-competes with BMS-936559 for binding. In one preferred embodiment, the cancer to be treated is MEL, RCC, squamous NSCLC, non-squamous NSCLC, CRC, castration-resistant prostate cancer, CRPC, HCC, squamous cell carcinoma of the head and neck, esophagus, ovary, gastrointestinal tract And breast carcinoma, and hematological malignancies.

  In one embodiment of the described method, the predetermined threshold value comprises: (a) tumor cells, (b) tumor infiltrating inflammatory cells, (c) in a test tissue sample expressing PD-L1 on the cell surface. A) Based on the proportion of specific tumor-infiltrating inflammatory cells, such as TILs or macrophages, or (d) a combination of tumor cells and tumor-infiltrating inflammatory cells. In one embodiment, the predetermined threshold value is a tumor cell that expresses at least 0.001% of the membrane PD-L1 as determined by IHC. In other embodiments, the predetermined threshold is a tumor cell that expresses at least 0.01%, preferably at least 0.1%, more preferably at least 1% of membrane PD-L1 as determined by IHC. It is. In one embodiment, the predetermined threshold is a tumor cell that expresses at least 5% of the membrane PD-L1 as determined by IHC. In one embodiment, the predetermined threshold is at least 0.01%, at least 0.1%, at least 1%, or at least 5% of a tumor cell that expresses membrane PD-L1 as determined by IHC. And / or a single tumor infiltrating inflammatory cell that expresses membrane PD-L1 as determined by IHC. In one other embodiment, the predetermined threshold value expresses at least 0.01%, at least 0.1%, at least 1%, or at least 5% of the membrane PD-L1 as determined by IHC. Tumor infiltrating inflammatory cells. In one other embodiment, the predetermined threshold value expresses at least 0.01%, at least 0.1%, at least 1%, or at least 5% of the membrane PD-L1 as determined by IHC. Tumor infiltrating lymphocytes. In one other embodiment, the predetermined threshold value expresses at least 0.01%, at least 0.1%, at least 1%, or at least 5% of the membrane PD-L1 as determined by IHC. Tumor infiltrating macrophages. In yet other embodiments, the predetermined threshold is a single tumor cell or a single tumor-infiltrating inflammatory cell that expresses at least PD-L1 of the membrane as determined by IHC. Preferably, PD-L1 expression is assayed by automated IHC using mAb 28-8 or 5H1 as the primary Ab.

  The application also provides (a) (i) optionally providing a test tissue sample from a plurality of subjects, wherein the test tissue sample comprises tumor cells and tumor infiltrating inflammatory cells; (ii) test tissue Assessing the percentage of cells expressing PD-L1 on the cell surface in the sample; and (iii) determining the percentage of cells in the test tissue sample from which the PD-L1 is to be expressed on the cell surface is predetermined. Selecting a subject as a candidate unsuitable for immunotherapy based on the assessment of being below the level, comprising administering to the subject an agent that inhibits an inhibitory immune regulator, eg, an anti-PD-1 Ab. Screening multiple subjects to identify subjects that are not suitable candidates for therapy; and (b) inhibiting an inhibitory immune regulator Agents, for example, comprises administering to a subject a selected standard therapeutic treatment other than anti -PD-1 Ab, provides a method for the treatment of a subject suffering from cancer.

  The application provides (a) (i) optionally, a test tissue sample from a plurality of subjects, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (ii) a test tissue sample Assessing the percentage of cells expressing PD-L1 on the cell surface in the medium; and (iii) determining the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample at a predetermined threshold level. Selecting a subject as a candidate suitable for immunotherapy with an agent that inhibits an inhibitory immune regulator, eg, anti-PD-1 Ab, based on the assessment of Screening a plurality of subjects to identify subjects that are suitable candidates; and (b) administering to the selected subjects a therapeutically effective amount of a composition comprising the agent. Including further provides methods for immunotherapy of a subject suffering from cancer.

  The application provides (a) (i) optionally, a test tissue sample from a plurality of subjects, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (ii) a test tissue sample Assessing the percentage of cells expressing PD-L1 on the cell surface in which the percentage of cells in a tissue sample expressing PD-L1 on the cell surface exceeds a predetermined threshold level; When the subject has been identified as a suitable candidate for anti-PD-1 Ab immunotherapy and the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample is below a predetermined threshold level, The subject is identified as a candidate that is not a suitable candidate for anti-PD-1 Ab immunotherapy, including multiple pairs to identify subjects that are suitable candidates for treatment. Screening for an elephant; and (b) a composition comprising a therapeutically effective amount of an agent that inhibits an inhibitory immune regulator, eg, an anti-PD-1 Ab, suitable for anti-PD-1 Ab immunotherapy. Administering to a subject identified as a candidate, or (c) administering a standard therapeutic agent other than the agent to a subject identified as not a suitable candidate for anti-PD-1 Ab immunotherapy. Further provided is a method for the treatment of a subject suffering from cancer, comprising:

  One aspect of the invention provides (a) optionally providing a test tissue sample obtained from a patient having a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; b) determining that the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample exceeds a predetermined threshold level; and (c) a therapeutically effective amount of inhibitory immunity based on the determination. A method for immunotherapy of a subject suffering from cancer, comprising administering to the subject a composition comprising a regulator inhibiting agent, for example, an anti-PD-1 Ab. Another aspect of the invention provides (a) optionally, providing a test tissue sample obtained from a patient having a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; b) determining that the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample is below a predetermined threshold level; and (c) inhibiting the inhibitory immune regulator based on the determination A method of treating a subject suffering from cancer, comprising administering to the subject a standard therapeutic agent other than an anti-PD-1 Ab, such as an anti-PD-1 Ab.

  Yet another aspect of the invention provides (a) optionally, providing a test tissue sample obtained from a patient having a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (B) determining the percentage of cells expressing PD-L1 on the cell surface in the test tissue sample; (c) anti-PD-1 Ab, the test tissue sample expressing PD-L1 on the cell surface Select an agent that inhibits an inhibitory immune regulator, such as an anti-PD-1 Ab, as a treatment for a subject based on the recognition that it is effective in a patient that contains a percentage of cells above a predetermined threshold level And d.) Administering to the subject a therapeutically effective amount of a composition comprising the agent, a method for immunotherapy of a subject with cancer. In other aspects, the application provides (a) optionally, a test tissue sample obtained from a patient with a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (B) determining the percentage of cells that express PD-L1 on the cell surface in the test tissue sample; (c) determining the percentage of cells that express PD-L1 on the cell surface in the test tissue sample. An agent that inhibits an inhibitory immune regulator as a treatment for a subject, eg, a standard therapeutic treatment other than an anti-PD-1 Ab, based on the recognition that the proportion is not effective in a patient below a predetermined threshold level There is provided a method for immunotherapy of a subject suffering from cancer, comprising selecting a drug; and (d) administering to the subject a standard therapeutic treatment.

  The present application also provides (a) assaying cells of a test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, to assess the percentage of cells in the test tissue sample expressing PD-L1; and (b) A) a subject inhibiting a therapeutically effective amount of an inhibitory immunoregulator, such as an anti-PD, based on the assessment that the percentage of cells expressing PD-L1 in the membrane exceeds a predetermined threshold level; There is provided a method of selecting an immunotherapy for a subject suffering from cancer, comprising selecting an immunotherapy comprising -1 Ab. The present application provides (a) assaying cells of a test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, to assess the percentage of cells in the test tissue sample expressing PD-L1; and (b) Based on the assessment that the percentage of cells expressing PD-L1 in the membrane is below a predetermined threshold level, the subject can be treated with an agent that inhibits an inhibitory immune regulator, eg, other than an anti-PD-1 Ab Further provided is a method of selecting a treatment for a subject afflicted with cancer, comprising selecting a standard therapeutic treatment.

  In addition, the present application provides a method of treating a subject suffering from a cancer comprising administering to the subject a composition comprising a therapeutically effective amount of an inhibitory immunoregulator, eg, an anti-PD-1 Ab. Wherein the subject has been selected based on the determination that the proportion of cells in a test tissue sample from a subject expressing PD-L1 exceeds a predetermined threshold level. , Wherein said test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells. The present application also provides a method for treating a subject suffering from cancer, comprising administering to the subject an agent that inhibits an inhibitory immune regulator, eg, a standard therapeutic treatment other than an anti-PD-1 Ab. Wherein the subject has been selected based on being determined such that the percentage of cells in a test tissue sample from the subject expressing PD-L1 is below a predetermined threshold level; The method provides that the sample comprises tumor cells and tumor infiltrating inflammatory cells.

  The present application provides (a) optionally, a test tissue sample obtained from a patient having a tissue cancer, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (b) cell surface Assaying a test tissue sample to determine the percentage of cells expressing PD-L1 above; (c) comparing the percentage of cells expressing PD-L1 on the cell surface with a predetermined threshold percentage And (d) selecting a patient for immunotherapy based on an assessment that the percentage of cells in the test tissue sample expressing surface PD-L1 exceeds a predetermined threshold level. Further provided are methods for selecting a cancer patient for immunotherapy with an agent that inhibits a sexual immune regulator, eg, an anti-PD-1 Ab.

  In any of the methods described herein, including a step for assessing PD-L1 expression, the test tissue sample can be a FFPE tissue sample, and the PD-L1 polypeptide on the cell surface has an anti- Detected by IHC using PD-L1 Ab, eg, mAb 28-8 or 5H1.

  In addition, any time the immunotherapy is selected or administered based on an assessment that the percentage of cells in the test tissue sample from the subject expresses PD-L1 at a level above a predetermined threshold level. In the method, a standard therapeutic treatment other than immunotherapy is selected or administered based on an assessment that the proportion of cells in the test tissue sample from the subject expresses PD-L1 at a level below a predetermined threshold level. If so, a complementary method of treatment could be performed.

  The present application provides (a) optionally a test tissue sample obtained from a patient having a cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (b) cell surface Assaying a test tissue sample to determine the percentage of cells expressing PD-L1 above; (c) comparing the percentage of cells expressing PD-L1 on the cell surface to a predetermined threshold. And (d) predicting the therapeutic effect of the agent, wherein the agent is effective in treating a patient when the percentage of cells expressing PD-L1 on the cell surface exceeds a threshold percentage Treating the cancer patient, including predicting that the percentage of cells expressing PD-L1 on the cell surface is less than the threshold percentage when the agent is predicted to be ineffective in treating the patient. For agents that inhibit the inhibitory immune regulator, for example, further provides a method for predicting the therapeutic effect of anti -PD-1 Ab.

  The application also provides (a) optionally, a test tissue sample obtained from a patient having cancer of the tissue, wherein the test tissue sample comprises tumor cells and tumor-infiltrating inflammatory cells; (b) cells Assaying the test tissue sample to determine the percentage of cells expressing PD-L1 on the surface; (c) determining the percentage of cells expressing PD-L1 on the cell surface with a predetermined threshold percentage. Comparing; and (d) determining an immunotherapeutic regimen comprising the agent based on a determination that the percentage of cells expressing PD-L1 on the cell surface exceeds a predetermined threshold percentage. Provided are methods for determining an immunotherapeutic regimen that includes an inhibitor of an inhibitory immune regulator, eg, an anti-PD-1 Ab, for treating a cancer patient.

Standard Therapeutic Therapy Some methods of treatment described herein involve administration of a standard therapeutic to a patient. As used herein, a "standard therapeutic treatment" is a drug or combination of drugs, radiation therapy (RT), surgery, or, where appropriate, recognized, accepted, and / or A treatment process that involves other medical interventions that are widely used for one type of patient, disease, or clinical situation. Standard treatments for various types of cancer are well known to those skilled in the art. For example, the National Comprehensive Cancer Information Network (NCCN), a partnership of 21 major cancer centers in the USA, provides detailed and up-to-date information on standard treatments for a wide variety of cancers (NCCN GUIDELINES®, 2013). The NCCN Standard Therapeutic Guidelines in Oncology (NCCN GUIDELINES®) is provided. As an example, standard therapeutic treatments for MEL, RCC and NSCLC are summarized below.

Melanoma MEL is a malignant tumor of melanocytes, a melanocyte producing cell found predominantly in the skin. Although less common than other skin cancers, it is the most dangerous skin cancer that, if not diagnosed early, will kill the majority (75%) of skin cancers. The incidence of MEL is increasing worldwide, in Caucasian populations, especially when people with low amounts of skin pigmentation are exposed to excessive UV exposure from the sun. The incidence is <10-20 per 100,000 population in Europe; 20-30 per 100,000 in USA; and per 100,000 in Australia where the highest incidence is observed. 50-60 people (Garbe et al., 2012). MEL accounts for about 5% of all new cases of cancer in the United States (US) and the incidence continues to increase by about 3% per year. This is the 2013 U.S.A. with 9,480 people associated with death. S. It is estimated that there are 76,690 new cases (Siegel et al. (2013)).

  Surgical resection is the main procedure in situ (stage 0) or early MEL (stage I-II). In general, the prognosis is good with 5-year survival of 90% or more for patients with localized disease and tumors with a thickness of 1.0 mm or less (NCCN GUIDELINES®, 2013-melanoma) ). If surgical resection is not suitable for in situ melanoma due to comorbidities or cosmetically sensitive tumor sites, topical imiquimod and radiation therapy are emerging as treatments, especially for malignant lentigo. Chemotherapeutic agents for treating MEL include dacarbazine, temozolomide and imatinib for melanoma with c-KIT mutation, high dose interleukin-2, and paclitaxel with or without carboplatin . However, these treatments have less than a 20% response rate in the first-line (1L) and second-line (2L) settings, with a modest success rate.

  In patients with localized melanoma greater than 1.0 mm thick, survival rates range from 50-90%. The likelihood of regional lymph node involvement increases with increasing tumor thickness. With stage III MEL (clinical positive nodules and / or in-transit disease), 5-year survival rates range from 20-70%. By far the most lethal is stage IV MEL with less than 10% long-term survival in patients with distant metastatic melanoma (NCCN GUIDELINES®, 2013-melanoma).

  A variety of treatments including excision, intralesional injection, laser ablation, radiation therapy and biochemotherapy (combination of chemotherapy and biological agents such as interferon-alpha and IL-2) for sites remote from the cancer are being studied However, the best treatment for metastatic MEL is not specified. The therapeutic perspective on metastatic MEL has recently seen a dramatic improvement in the development of new drugs, such as Vemurafenib and Ipilimumab. Vemurafenib specifically inhibits signaling by BRAF, a mutated intracellular kinase that is present in about 50% of patients with metastatic MEL. In clinical trials, vemurafenib was estimated at dacarbazine in BRAF mutant-positive patients only at 1.6 months putative progression free survival (PFS) versus 5.3 months at putative progression free survival (PFS), and at dacarbazine. The central OS in Vemurafenib is given to the central OS in 5.6-7.8 in 15.9 (Chapman et al., 2011; Sosman et al., 2012). Ipilimumab is a HuMAb that inhibits the immune checkpoint receptor, CTLA-4, and thereby stimulates a T cell immune response. Ipilimumab with or without the glycoprotein 100 (gp100) peptide vaccine is 6.4 months in patients with previously treated metastatic MEL, in patients receiving gp100 alone Improved to the central OS in 10.0-10.10 months compared to the central OS (Hodi et al., 2010). In addition to these two drugs, no other drugs have demonstrated OS benefit in phase 3 randomized trials. Dacarbazine has been approved by the FDA and EMA for the treatment of metastatic MEL with a reported objective response rate of 5-20% and a central OS of about 6.4 months, but these responses are short-lived. is there. Other drugs, such as temozolomide and fotemustine, did not cause a significant improvement in survival compared to dacarbazine. IL-2 has also been associated with a 15-20% response rate, including a 4-6% complete response, which can be sustained, but with significant toxicity including hypotension, cardiac arrhythmias and pulmonary edema. As approved by the FDA for the treatment of metastatic MEL. Further details of standard therapeutic treatments for melanoma are provided by Garbe et al. (2012) and NCCN GUIDELINES®, 2013-Melanoma. Despite recent approval of ipilimumab and vemurafenib for advanced MEL, patients who have progressed (depending on BRAF status) in anti-CTLA-4 therapy and BRAF inhibitors, or previously untreated, unresectable There is still a significant unmet need for patients with metastatic BRAF wild-type MEL. The 5-year survival rate for late MEL is currently only 15%.

Renal cell carcinoma RCC is the most common type of renal cancer in adults, with approximately 90% of kidney tumors being 80-90% clear cell tumors (NCCN GUIDELINES®, 2013-renal cancer) ). It is also the most deadly of all genitourinary tumors. An estimated 65,150 patients have been diagnosed with kidney cancer and 13,680 have died from the disease in 2013 in the United States (Siegel et al. (2013)).

  In clinically localized RCC (stages IA and IB), surgical resection, including radical nephrectomy and nephrectomy, is an effective treatment. Partial nephrectomy is generally not appropriate for patients with locally advanced tumors (stages II and III), in which case a radical nephrectomy is preferred. If the tumor is confined to the renal parenchyma, the 5-year survival rate is 60-70%, which is much lower in stage IV disease when metastases have spread. Stage IV RCCs are relatively resistant to RT and chemotherapy, and patients can benefit from surgery, but surgical renal removal with potential for tumor shrinkage prior to systemic therapy Recommended for patients with resectable primary and multiple resectable metastases.

  Until recently, the cytokines IL-2 and IFNα were the only active systemic treatments for advanced or metastatic RCC, both providing 5-27% of the reported ORR. However, due to the limited clinical benefit and substantial toxicity profile of each of these agents, recent targeted agents have been widely replaced by cytokines in the treatment of advanced or metastatic renal cell carcinoma. Recognition of the importance of hypoxia inducible factor alpha (HIFα) signaling in the pathogenesis of clear cell RCC has been shown in IL and 2L treatment to be two classes of targeted therapies, anti-angiogenic tyrosine kinase inhibitors (TKIs) and mammalian targets Has caused extensive testing of rapamycin (mTOR) inhibitors (Mulders, 2009). Targeting angiogenesis is rational because constitutive HIFα activation causes up-regulation or activation of several proteins, including vascular endothelial cell growth factor (VEGF), which can later cause tumor growth and neovascularization. is there. Targeting the mTOR pathway is important because activation of the upstream PI3K / Akt / mTOR signaling pathway is one way in which constitutive HIFα activation or up-regulation occurs (Mulders, 2009). Agents that target angiogenesis include VEGF-receptor (VEGFr) TKIs (eg, sorafenib, sunitinib, pazopanib, axitinib, and tibozanib) and VEGF-binding mAbs (eg, bevacizumab), but agents that target the mTOR pathway Include mTOR inhibitors (eg, everolimus and temsirolimus) (Mulders, 2009; NCCN GUIDELINES®, 2013-kidney cancer). However, a large number of patients develop tolerance, and OS improvement has been shown only in one phase 3 trial in high-risk patients: temsirolimus has been shown to increase OS in patients with advanced RCC compared to IFNα. Showed a statistically significant benefit (Hudes et al., 2007). Everolimus also has no OS improvement over placebo, but has demonstrated a 2.1 month improvement in central PFS (Motzer et al., 2008). Of the five approved anti-angiogenic agents (sorafenib, sunitinib, bevacizumab, pazopanib and axitinib) and two approved mTOR inhibitors (temsirolimus, everolimus), only everolimus has failed after treatment with anti-angiogenic therapy Has been specifically approved for use. In the United States, everolimus is indicated for the treatment of progressive RCC after failure of treatment with sunitinib or sorafenib, and in Europe, everolimus is more widely indicated for patients with advanced RCC. The disease progresses during or after treatment with VEGF-targeted therapy.

Non-Small Cell Lung Cancer NSCLC is the leading cause of cancer death in the United States and around the world, beyond the combination of breast, colon and prostate cancer. In the United States, an estimated 228,190 new cases of lung and bronchi are diagnosed in the United States, and the disease causes approximately 159,480 deaths (Siegel et al., 2013). The majority of patients (about 78%) are diagnosed with advanced / recurrent or metastatic disease. Metastasis from lung cancer to the adrenal gland commonly occurs, and about 33% of patients have such metastasis. NSCLC treatment is gradually improving OS, but benefit has reached a plateau (the central OS for terminally ill patients is just one year). Progression after 1L treatment occurs in almost all of these subjects, with a 5-year survival rate of only 3.6% in the refractory setting. From 2005 to 2009, the overall 5-year relative survival rate for lung cancer in the United States was 15.9% (NCCN GUIDELINES®, 2013-non-small cell lung cancer).

  Surgery, RT and chemotherapy are three commonly used modalities for treating NSCLC patients. As a class, NSCLC is relatively less responsive to chemotherapy and RT compared to small cell carcinoma. In general, in patients with stage I or II disease, surgical resection, together with increasingly used chemotherapy before and after surgery, offers the best opportunity for care. RT may also be used as adjuvant therapy for patients with resectable NSCLC, initial local treatment, or palliative therapy for patients with non-curable NSCLC.

  Patients with stage IV disease with good general condition (PS) benefit from chemotherapy. A number of drugs are useful for Stage IV NSCLC, including platinum drugs (eg, cisplatin, carboplatin), taxane drugs (eg, paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, and gemcitabine. is there. Combinations that use many of these drugs produce 30% to 40% one-year survival rates and are superior to single drugs. Certain targeted therapies have also been developed for the treatment of advanced lung cancer. For example, bevacizumab (AVASTIN®) is a mAb that blocks vascular endothelial cell growth factor A (VEGF-A). Erlotinib (Tarceva®) is a small molecule of the epidermal growth factor receptor (EGFR) TKI. Crizotinib (XALKORI®) is a small molecule TKI that targets ALK and MET and is used to treat NSCLC in patients with a mutated ALK fusion gene. Cetuximab (Arbitux®) is a mAb that targets the EGFR.

  There are specific unmet needs for patients with squamous NSCLC (representing up to 25% of all NSCLC) due to the small treatment options after 1L treatment. Monotherapy is the standard treatment after progression with dual chemotherapy with platinum (Pt dual), causing a central OS of about July. Erlotinib may also be used less frequently, but docetaxel maintains benchmark treatment in this line of therapy. Pemetrexed has also been shown to produce clinically equivalent efficacy outcomes with significantly fewer side effects than docetaxel in 2L treatment of patients with advanced NSCLC (Hanna et al., 2004). There are currently no treatments approved for use in lung cancer beyond the 3L setting. Pemetrexed and bevacizumab have not been approved in squamous NSCLC and molecular targeted therapy has limited applications. The unmet need in advanced lung cancer is the recent failure of Oncothreon and Merck KgaA's STIMUVAX® to improve OS in Phase 3 trials, ArQuule and Daiichi Sankyo to meet survival endpoints. Inability of the c-Met kinase inhibitor tivantinib, failure of Eli Lilly's ALIMTA (R) in combination with Roche's AVASTIN (R) to improve OS in late studies, and the small molecule VEGF in late studies Exacerbated by the failure of Amgen and Takeda to meet clinical endpoints with the -R antagonist motesanib.

  The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all documents referred to throughout this application are hereby incorporated by reference.

Example 1
Cross-competition between anti-PD-1 HuMAbs for binding to CHO cells expressing human PD-1 Chinese hamster ovary (CHO) transfected to express human PD-1 (CHO / PD-1 cells) Cells were incubated with 10 μg / ml anti-PD-1 HuMAb 5C4 or the Fab fragment of the human IgG1 (hIgG1) isotype control Ab at 4 ° C. for 30 minutes and anti-PD-1 at a concentration of 0.2 μg / ml. HuMAbs 2D3, 7D3 or 4H1 were added. Binding of 4H1, 2D3 or 7D3 to CHO / PD-1 cells was detected by fluorescein isothiocyanate (FITC) conjugated goat anti-hIgG, Fc-gamma specific Ab. In the case of a cross-competition assay with 5C4 and 17D8, CHO / PD-1 cells were incubated with all molecules of 5C4 and FITC-labeled 17D8 was added. Binding of 2D3, 7D3, 4H1 or 17D8 to CHO / PD-1 cells was measured by flow cytometry analysis using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA).

  The results are shown in FIG. The data indicate that the 5C4 Fab fragment substantially blocks the binding of mAb 5C4 itself, as well as the binding of 2D3, 7D3 (FIG. 1A) and 4H1 (FIG. 1B), as measured by the mean fluorescence intensity (MFI) of the staining. 5C4 shows that all mAbs substantially blocked the binding of 17D8 (FIG. 1C) to CHO / PD-1 cells.

Example 2
Cross-competition between anti-PD-L1 HuMAbs for binding to CHO cells expressing human PD-L1 CHO cells transfected to express hPD-L1 (CHO / PD-L1 cells) at 4 ° C. In 20 min, 10 μg / ml of 10 unconjugated human anti-PD-L1 mAbs (5F8, 7H1, 10H10, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7 and 13G4) or human IgG1 (hIgG1), respectively. ) Incubated with isotype control Ab. FITC-conjugate 10H10 (A), 3G10 (B), 10A5 (C), 11E6 (D), 12A4 (E) or 13G4 (F), unbound, washout of unconjugated Ab Without 0.09 μg / ml (B, D), 0.27 μg / ml (A, C), 0.91 μg / ml (F), or 2.73 μg / ml (E) for an additional 20 minutes at 4 ° C. Was added to the cells to a final concentration of. Different amounts of different FITC-conjugated HuMAbs were used due to differences in binding efficiencies after labeling, and the optimal amount of these FITC-conjugated HuMAbs was determined for binding to CHO / PD-L1 cells. It was previously determined by dose titration analysis. Binding of FITC-conjugate 10H10, 3G10, 10A5, 11E6, 12A4 or 13G4 to CHO / PD-L1 cells was measured by flow cytometry.

  The results are shown in FIG. The binding of labeled 10H10 was partially blocked by 10A5, 11E6 and 13G4, but was substantially blocked only by itself (FIG. 2A). Conversely, 10H10 substantially blocked its own binding to CHO / PD-L1 cells. Each of the anti-PD-L1 HuMAbs 5F8, 7H1, 1B12, 3G10, 10A5, 11E6, 12A4, 12B7 and 13G4, as measured by MFI, labeled mAb 3G10 on CHO / PD-L1 cells (FIG. 2B). , 10A5 (FIG. 2C), 11E6 (FIG. 2D), 12A4 (FIG. 2E) and 13G4 (FIG. 2F) are substantially blocked, whereas mAbs 5F8 and 13G4 reduce binding of the labeled mAb to a lesser extent. Generally blocked.

Example 3
Cross-competition between anti-PD-L1 mAbs for binding to ovarian carcinoma cells expressing human PD-L1 Anti-PD-L1 HuMAbs 5F8, 12B7, 3G10, 1B12, 13G4, 10H10, 10A5 and 12A4, and human IgG1 ( huIgG1) isotype control Abs were serially diluted from 10 μg / ml and incubated with hPD-L1 expressing ES-2 ovarian carcinoma cells for 20 minutes at 4 ° C. Without washing, biotinylated-12A4 Ab was added to a final concentration of 0.4 μg / ml at 4 ° C. for an additional 20 minutes. After washing, bound biotin-12A4 was detected using a fluorescent streptavidin-PE secondary reagent and measured by flow cytometry. FIG. 3 shows the fluorescence of bound biotin-12A4 plotted against the concentration of unlabeled hPD-L1 HuMAb. Biotin-12A4 binding to ES-2 cells was substantially blocked by 12A4 itself and 1B12 and 12B7, and moderately significantly blocked by mAbs 5F8, 10A5, 13G4 and 3G10, but blocked by mAb 10H10. Was not done.

Example 4
Design of a Phase 1 Clinical Trial of Anti-PD-1 Ab Phase 1 trials were designed to evaluate the safety, antitumor activity and pharmacokinetics of anti-PD-1 in patients with selected advanced solid tumors went. BMS-936558, a human anti-PD-1 mAb (also referred to herein as nivolumab or 5C4 in U.S. Patent No. 8,008,449), was administered intravenously every two weeks of an eight week treatment cycle each. It was administered as an infusion. Tumor status was re-evaluated after each cycle. Patients continued treatment for up to 2 years (12 cycles) until they experienced complete remission, unacceptable toxicity, disease progression, or withdrew consent. In other clinically stable patients, trial treatment was continued beyond the apparent initial disease progression until further progression appeared, as recommended by the proposed immune response criteria (Wolchok et al., 2009) Was. Patients with stable disease (SD) or continuous objective response (OR: complete response [CR] or partial response [PR]) at the end of treatment are followed for one year and re-treatment for another year in the case of progression Offered.

Dose escalation Patients with advanced melanoma (MEL), non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), castration resistant prostate cancer (CRPC) and colorectal cancer (CRC) are enrolled (enroll ) Was eligible. A cohort of 3-6 patients per dose level was enrolled consecutively at 1.0, 3.0 and 10.0 mg / kg. Dose escalation was performed with dose limiting toxicity in <1/3 patients when a minimum of 3 patients completed the safety evaluation period (56 days) at the given dose level. Intra-patient dose escalation was not allowed.

Cohort expansion Maximum tolerated dose (MTD) was not reached. Initially, each of five expanded cohorts of approximately 16 patients was enrolled at 10 mg / kg for MEL, NSCLC, RCC, CRPC and CRC. Based on the initial signal of activity and the 6.5 month break for the next protocol modification, an additional expansion cohort of approximately 16 patients was given MEL (1.0 and 3.0 mg / kg at In addition, cohorts randomized to 0.1, 0.3 or 1.0 mg / kg, NSCLC (squamous or non-squamous tissue cohort randomized to 1, 3 or 10 mg / kg), and RCC ( 1.0 mg / kg). Intra-patient dose escalation to 1 mg / kg was allowed for MEL patients with progressive disease after receiving 0.1 or 0.3 mg / kg.

Patients Eligible patients are advanced solid tumors; age> 18 years; life expectancy> 12 weeks; general condition of the US East Coast Cooperative Oncology Group of ≦ 2; solid tumor response assessment criteria (RECIST), revised Measurable disease with v1.0 plus (see Topalian et al., 2012b); demonstrated adequate hematology, liver and kidney function; received 1-5 prior to systemic treatment regimen. Patients with stable treated brain metastases were enrolled. Exclusion criteria include history of chronic autoimmune disease, previous treatment with Abs that regulate T cells (eg, anti-CTLA-4, anti-PD-1, anti-PD-L1), immunosuppressive drug treatment And chronic infections (eg, HIV, hepatitis B or C).

  A total of 296 patients with advanced solid tumors, including MEL (n = 104), NSCLC (n = 122), RCC (n = 34), CRPC (n = 17), and CRC (n = 19), Up to February 2012, treated with BMS-936558 for 40 months. Patients with non-small cell lung cancer (n = 129), melanoma (n = 107), RCC (n = 34), CRPC (n = 17), and CRC (n = 19) by March 2013 306 patients were treated with BMS-936558 from October 2008 to January 2012, and all were observed for a minimum of one year. Two patients did not receive a complete cycle of treatment and did not appear to be able to assess response. The median age was 63 years (range 29-85). The ECOG behavioral score was 0 or 1 in 98% of patients. The majority of patients were well pre-treated, with 47% receiving at least 3 prior to the regimen. Notable previous treatments were immunotherapy (64%) and B-RAF inhibitor (8%) in MEL patients; chemotherapy with platinum (94%) and tyrosine kinase inhibitors (TKI, 34%) in NSCLC patients. And nephrectomy (94%), immunotherapy (59%) and anti-angiogenic treatment (74%) in RCC patients. Baseline characteristics of all treated populations (N = 306) were similar to those of the efficacy population (evaluable patients for response, N = 270). Details of patient preparation are provided in Topalian et al. (2012b).

Statistical Analysis Baseline characteristics and adverse events for all treated patients (N = 306) and efficacy results for 270 patients with lung, melanoma, and kidney cancer in March 2013 are reported. The pharmacokinetic and molecular marker population consisted of treated patients with data available for preliminary analysis in February 2012. The efficacy population consisted of patients evaluable for response to starting treatment at least eight months prior to the day of analysis. Tumor measurements were collected after each treatment cycle (4 times) by the investigator. Individual best objective responses based on tumor measurements were evaluated by the sponsor with a modified RECIST v1.0. Objective response was confirmed by at least one continuous tumor assessment. Objective response and stable disease rates were estimated with confidence intervals using the Cropper-Pearson method. Time-to-event endpoints, including PFS, OS, viability, and response duration, were estimated using the Kaplan-Meier method. The AE was coded using the International Medical Glossary of Terms (MedDRA), version 15.1. A possible immunological etiology, also referred to as "immune-related adverse events" in "AEs of particular interest" (AEOSI), defined as adverse events requiring frequent monitoring and / or unique interventions Select adverse events were identified using a predefined list of MeDRA terms. Individual best ORs were from investigator-reported data with modified RECIST v1.0. OR was confirmed by at least one continuous tumor assessment and the OR rate (ORR = {[CR + PR] {n} × 100) was calculated.

Example 5
Safety Assessments for Patients Treated with Anti-PD-1 Antibody Safety assessments, including laboratory and laboratory evaluations, were performed at baseline and at regular intervals up to 100 days after the last dose of drug in all treated patients. went. AE severity was graded based on the NCI Adverse Event Common Terms Standard (NCI CTCAE), v3.0. Computed tomography (CT) or magnetic resonance imaging was performed at baseline and after each treatment cycle for tumor assessment.

  The MTD was not defined beyond the dose of BMS-936558 tested for this study and was up to the highest planned dose of 10 mg / kg. Over 90% of the relative BMS-936558 dose intensities were achieved in 87% of the patients (see Topalian et al., 2012b; Topalian et al., 2013 for details). The AE was coded using the International Medical Glossary of Terms (MedDRA), version 14.1. AEOSI was identified using a pre-defined list of MeDRA terms. Fifteen (5%) of the 296 patients discontinued treatment with BMS-936558-related AEs. In a preliminary analysis in February 2012, 62 (21%) patients died, and by March 2013, 195 patients (64%) died of disease progression, the most common cause of death ( Topalian et al., 2012b; Topalian et al., 2013).

  The most common adverse events, regardless of causality, included fatigue, loss of appetite, diarrhea, vomiting, cough, dyspnea, constipation, vomiting, rash, fever and pruritus (Topalian et al., 2012b; Topalian et al., 2013). Common BMS-936558-related AEs included fatigue, rash, diarrhea, decreased appetite and vomiting. The majority of events were of low grade, with grade 3-4 drug-related AEs observed in 41 of 296 (14%) patients. Treatment-related AEs (any grade) were observed in 230 of 306 (75%) patients, the most common being fatigue, rash, diarrhea and pruritus (Topalian et al., 2013). The range, frequency, and severity of BMS-936558-related AEs were generally similar across the treated dose levels, with no apparent association with the duration of treatment. 52 of the 306 patients (17%) experienced grade 3-4 treatment-related adverse events, including fatigue (2%), pneumonia, lymphopenia, diarrhea, abdominal pain and hypophosphatemia (1% each). %) Was the most common. Serious treatment-related adverse events occurred in 42 of 306 patients (14%) (Topalian et al., 2013).

  Drug-related AEOSIs with a potential immune-related etiology were classified by organ damage to provide a more accurate estimate of frequency. In particular, it included pneumonia, vitiligo, colitis, hepatitis, pituititis and thyroiditis. Either grade of treatment-related selected adverse events were observed in 140 of 306 patients (46%), the most common being rash (15%), diarrhea (13%) and pruritus (11%) (Table 2). Grade 3-4 treatment-related selection events were seen in 19 patients (6%).

  Of the 230 patients with drug-related adverse events, 52 patients (23%) required management with systemic glucocorticoids and / or other immunosuppressants. Liver or gastrointestinal AEOSI was administered with treatment obstruction, with corticosteroid administration as needed. Of the patients treated to date, these AEs were reversible in all cases. Endocrine AEOSI was managed with replacement therapy. Thirty-two (11%) patients out of 306 discontinued treatment due to treatment-related adverse events. At the discretion of the treating physician, the patient successfully resumed treatment with BMS-936558. Twenty-one (40%) were able to resume after toxicity was resolved.

  Drug-associated pneumonia (any grade) occurred in 12 (4%) of the 306 patients (Topalian et al., 2013). Clinical presentation ranged from radiological abnormalities in asymptomatic patients to symptoms associated with progressive, diffuse pulmonary infiltration (cough, fever, dyspnea). Grade 3-4 pneumonia developed in 4 patients (1%), of which 3 were fatal (2 NSCLC patients, 1 CRC). No clear association between pneumonia incidence and tumor type, dose level, or number of doses given was not noticed. In 9 of 12 patients, pneumonia was reversible with treatment discontinuation and / or immunosuppression (glucocorticoid, infliximab, mycophenolic acid). After the latest pneumonia-related death in November 2011, 79 patients continued to receive nivolumab (central 29 weeks, range 2-69 weeks) without further death from this or other treatment-related causes .

Example 6
Pharmacokinetic / Pharmacokinetic Analysis for Anti-PD-1 Antibody For pharmacokinetic (PK) analysis, serial blood samples were collected and serum concentrations of BMS-936558 were quantified using ELISA. For pharmacokinetic (PD) analysis, peripheral blood mononuclear cells were isolated from patients at baseline and after cycle 1, and PD-1 receptor occupancy by circulating CD3 + T-cells by BMS-936558 by flow cytometry ( RO) was estimated (Brahmer et al., 2010).

The maximum concentration of BMS-936558 was observed at the central T _max 1-4 hours after the start of the injection. The PK of BMS-936558 was linear with a dose proportional increase in C _max and AUC _{(0-14d) over} the dose range of 0.1-10 mg / kg (n = 35). BMS-936558 PD was assessed by PD-1 RO on circulating T-cells. PBMC from 65 MEL patients treated with one cycle of BMS-936558 were 0.1-10 mg / kg biweekly, circulating CD3 ^{+ with} BMS-936558 ranging from 64% -70%. Demonstrated central occupancy of the PD-1 molecule on T-cells (see Topalian et al., 2012b for details).

Example 7
Anti-tumor efficacy as demonstrated by anti-PD-1 antibody Data analyzed in February 2012 Clinical anti-tumor activity was observed at all BMS-936558 doses tested. OR (confirmed CR or PR) in a significant proportion of patients with NSCLC, MEL and RCC (Tables 1 and 2; FIG. 4) and for metastatic disease involving liver, lung, lymph nodes and bone It was observed at various sites (FIGS. 5-7 and not shown). Tumor regression was followed by conventional as well as "immune-related" patterns of response, such as a prolonged decrease in tumor burden in the presence of new lesions. Individual best overall responses were obtained from investigator-reported data with modified RECIST v1.0. OR was confirmed by at least one continuous tumor assessment. At the time of data analysis, two patients with NSCLC treated at 10 mg / kg had an unconfirmed response and eight additional patients (with MEL, NSCLC or RCC) had a baseline in the presence of new lesions It had a sustained reduction in the target lesion (ie, an "immune-related" response pattern). These patients were not classified as responders for the purpose of calculating the OR rate. Stabilization of anti-tumor response and / or long-term disease was observed in patients regardless of previous treatment received (Summary of progression-free time for patients with OR and SD in Supplementary Appendix 4 of Topalian et al., 2012b) ,reference).

  In NSCLC patients, 14 ORs were observed at BMS-936558 doses of 1, 3, or 10 mg / kg, respectively, with 6%, 32%, and 18% response rates. OR was observed across NSCLC tissue: 6 responders (33%) with 18 squamous epithelia, 7 responders (13%) with 56 non-squamous epithelia, and one out of two unknown. Prior to data analysis, a total of 14 patients with OR started treatment> 24 weeks, of which 8 had a response period> 24 weeks (Table 1). Stable disease (SD) lasting ≧ 24 weeks was observed in 5 (7%) of all NSCLC patients with non-squamous epithelium. In MEL patients, 26 ORs were observed at doses ranging from 0.1-10 mg / kg, with response rates ranging from 19% -41% depending on the dose level. At a dose level of 3 mg / kg, OR was noticed in 7 out of 17 (41%) patients. Seventeen of the 26 MEL patients who achieved OR started treatment> 1 year prior to data analysis, and 13 of these had an OR period> 1 year (yr). The remaining eight patients with OR had a study <1 year, and six had responses ranging from 1.9-5.6 months. SD lasting ≧ 24 weeks was observed in 6 (6%) patients. In RCC patients, the OR was 4 (24%) of 17 patients treated with a 1 mg / kg dose of BMS-936558 and 5 (31%) of 16 patients treated with 10 mg / kg. ). Of the 8 RCC patients with OR who started treatment> 1 year prior to data analysis, 5 (63%) had OR duration> 1 yr. SD lasting ≧ 24 weeks was observed in an additional 9 (27%) patients.

Table 1. Efficacy Population of Data Assessed by February 2012 ^* Clinical Activity of BMS-936558 in (N = 236) ^†

^* Efficacy population consisted of patients whose treatment was evaluable for response initiated at least 8 months prior to data analysis in February 2012, with disease measurable at baseline and one of the following: At least one treatment scan or clinical evidence of progression or death.
^† CR complete response, MEL melanoma, NSCLC non-small cell lung cancer, ORR is objective response rate, PFSR the progression-free survival, PR partial response, RCC renal cell carcinoma, SD stable disease, n indicates the number of patients.
^The {objective response rate ({[CR + PR] {n} × 100) "is calculated based on responses identified with confidence intervals calculated using the Cropper-Pearson method. Individual patient responses were determined by RECIST v1.0 with modifications (see Topalian et al., 2012b).
^§ Progression-free survival was the percentage of patients that survived 24 weeks as calculated by Kaplan-Meier methodology with confidence intervals using the Greenwood method.
^¶ One CR.
** One NSCLC patient treated at the 3 mg / kg dose level had an initial assessment of progressive disease, then had a PR, and was classified as a responder.

Table 2. Duration of objective response to BMS-936558 ^* assessed by February 2012

* MEL indicates melanoma, NA indicates not applicable, NSCLC indicates non-small cell lung cancer, and RCC indicates renal cell carcinoma.
^時間 Time to first progression, time to confirmed progression, death, or, for censored data, to last tumor assessment.
^‡ 1 patients, treated beyond the first evaluation of progressive disease, then have a PR; patient is classified as responders for the purpose of calculating the reaction rate by RECIST v1.0 However, it was not suitable for calculating the duration of the response.

Analyzed data for March 2013 Objective response was observed in patients with NSCLC (17%), MEL (31%) and RCC (29%), but not in patients with CRC or CRPC Was. Responses were observed across all nivolumab doses tested; response rates in NSCLC patients receiving 1 mg / kg vs. 3 or 10 mg / kg appeared to be decreasing (3% vs. 24%, respectively). And 20%) (Tables 3 and 4). Thirteen of the 270 patients (4.8%) with responding tissues had an unconventional response pattern that did not meet RECIST criteria (eg, the presence of new lesions or regression after initial progression) (Persistent reduction in target lesions) (Wolchok et al., 2009). Additional patients showed SD for 24 weeks or longer (10% NSCLC, 7% MEL, 27% RCC). Sustained survival, reflected by 1 and 2 year breakthrough OS rates, was noticed in each of the responding populations as follows: NSCLC, 42% and 14%; MEL, 62% and 43%; and RCC, 70% and 50% (Table 3). Median 9.6 months for lung cancer (9.2 and 10.1 months for squamous and non-squamous NSCLC tissue respectively), 16.8 months for MEL, and 22 months or more for RCC OS was observed; central PFS was 2.3 months in NSCLC, 3.7 months in MEL, and 7.3 months in RCC; and central response periods were 74, 104 and 56 weeks, respectively. (Table 4). Of the 16 responders who discontinued treatment for reasons other than disease progression and were followed for at least 24 weeks, 13 (81%) responded at the time of analysis (FIG. 8).

Table 3. Clinical activity of nivolumab in the efficacy population assessed in March 2013 (N = 306) ^*

１人のＣＲは黒色腫と示され、１人のＣＲは腎臓癌と示された。
^＃中央全生存は、この試験において腎臓癌を有する患者の範囲で死までの最も長い時間である、２２月で到達しなかった。
＾ＮＥ、評価できない。 * No objective response was seen in 19 patients with colorectal cancer or 17 patients with castration-resistant prostate cancer.
^† objective response rate ({[CR + PR] ÷ n} × 100) is calculated based on the responses observed in confidence intervals are calculated using Clopper-Pearson method. Individual patient responses were determined by RECIST v1.0 with modifications (see Method S1 and test protocol, NEJM.org).
^時間 Time to first confirmed response, time to confirmed progress, death, or for censored data (indicated by “+”), to last tumor assessment.
** Of 129 patients with non-small cell lung cancer, one had unknown tissue and did not show an objective response. Other patients had squamous or non-squamous epithelium as indicated.
^§ NR, not reached; The point at which the responder's chance of progressing to less than 50% was not reached due to an insufficient number of events and / or follow-up.
^¶ Inadequate tracking period.

One CR was designated as melanoma and one CR was designated as renal cancer.
^# Median overall survival was not reached at 22 months, the longest time to death in the range of patients with renal cancer in this study.
＾ NE, cannot be evaluated.

Table 4. Nivolumab clinical activity by dose level evaluated in March 2013

* The objective response rate ({[CR + PR] {n} × 100) is calculated based on the responses identified in the confidence intervals calculated using the Cropper-Pearson method. Individual patient responses were determined by RECIST v1.0 with modifications (see Method S1 and test protocol, NEJM.org).
^時間 Time to first confirmed response, time to confirmed progress, death, or for censored data (indicated by “+”), to last tumor assessment.
^‡ NR, not reached; The point at which responders were less than 50% likely to progress was not reached due to an insufficient number of events and / or follow-up.
^§ One CR was indicated as melanoma and one CR was indicated as renal cancer.
^¶ Five patients with tumor progression ^escalated from 0.1 to 1.0 mg / kg, and six escalated from 0.3 to 1.0 mg / kg. These patients did not respond to treatment.
^# Median overall survival was not reached at 22 months, the longest time to death in the range of patients with renal cancer in this study.
＾ NE, cannot be evaluated.

Example 8
Correlation Between Membrane PD-L1 Expression and Anti-PD-1 Response IHC staining of PD-L1 was determined by the mouse anti-human PD-L1 mAb 5H1 (Taube et al., 2012; Supp. Materials) in a standard IHC protocol (Taube et al., 2012; Supp. Materials). Dong et al., 2002) in pretreated formalin-fixed paraffin-embedded (FFPE) tumor specimens. Briefly, 5 μm-FFPE sections mounted on glass slides are deparaffinized in xylene and antigen retrieval using Tris-EDTA buffer at 120 ° C. for 10 minutes at pH 9.0 in an antigen stimulator (Biocare Medical). Was done. Endogenous peroxidase, biotin and protein are blocked (CAS system K1500, Dako; Avidin / biotin Blocking Kit, SP-2001, Vector Laboratories; Serotec Block ACE), primary 5H1 Ab is added at a concentration of 2 μg / ml and at 4 ° C. Incubated for 20 hours. Secondary Ab (biotinylated anti-mouse IgG1, 553441 BD) was applied at a concentration of 1 μg / ml for 30 minutes at room temperature (RT). The signal was then generated by amplification according to the manufacturer's protocol (CAS system K1500, Dako). Sections were counterstained with hematoxylin, dehydrated in ethanol, cleared in xylene, and coverslips applied.

  The percentage of tumor cells showing cell surface staining for PD-L1 was scored by two independent pathologists who obscure the treatment results. PD-L1 positivity was defined per sample by a 5% expression threshold (Taube et al., 2012; Thompson et al., 2006), in the case of multiple samples, when the sample met this criterion. Fisher's exact test was applied to assess the association between PD-L1 expression and OR. Note, however, that this analysis was based, in part, on any biopsy from a non-random subset of the population, and the test of the statistical hypothesis was not pre-specified.

Sixty-one pretreated tumor specimens from 42 patients (18 MEL, 10 NSCLC, 7 CRC, 5 RCC, and 2 CRPC) were analyzed for tumor cell surface PD-L1 expression (FIG. 8). ). Biopsy specimens from 25 to 42 patients were positive for PD-L1 expression by IHC. Fisher's exact test was applied to assess the association between PD-L1 expression and OR in a post-hoc analysis. Of the 42 surface-PD-L1 ⁺ patients, 9 (36%) achieved OR, whereas 17 patients with PD-L1 ^- tumor did not achieve OR (FIG. 8A). . Thus, in a subset of patients, cell surface expression of PD-L1 on tumor cells in pre-treatment biopsies is associated with an increased OR rate in patients treated with BMS-936558, but is demonstrated by PD-L1 -Patients with negative tumors did not experience OR. These data indicate that tumor PD-L1 expression is a molecular marker that can allow patient selection for anti-PD-1 immunotherapy.

Example 9
Isolation of Rabbit mAbs Directing Membrane hPD-L1 Antigen in FFPE Tissues Rabbit Abs against human PD-L1 polypeptide use recombinant human PD-L1 fusion protein by Epitomics, Inc. (Burlingame, Calif.). Prepared by immunization of rabbits. Antiserum titers were evaluated using a standard direct ELISA with hPD-L1 antigen and using a cell ELISA using transfected cells that overexpress hPD-L1. These Abs were also screened for their ability to bind to PD-L1 by IHC assay on FFPE tissue sections. The rabbit with the highest Ab titer was selected for splenectomy. Lymphocytes isolated from the spleen were fused to myeloma cells in 40x96-well plates and screened by ELISA for immune PD-L1 antigen and by cell ELISA for cells overexpressing hPD-L1. Positive clones were expanded into 24-well plates and confirmatory screening was performed by direct ELISA and cell ELISA. Supernatants (sups) of clones that were specific for the screening antigen were rescreened by IHC.

  A set of mouse anti-hPD-L1 mAbs was also produced by immunization of mice using a protocol similar to that described above for rabbit mAbs.

  Of a total of 185 multiclones from both rabbit and mouse immunizations screened, only 10 rabbit multiclones Ab specifically detected the membrane morphology of hPD-L1. The purified mouse subclone was not found to specifically detect cell surface hPD-L1. 60 subclones from the top 5 rabbit multiclones (designated numbers 13, 20, 28, 29 and 49, each containing 12 subclones) were first screened by IHC on FFPE low density tissue microarray (TMA). Then, confirmation and specificity verification were performed on the limited 25 subclones. Rabbit IgG was used as a negative isotype control and mAb 5H1 (Dong et al., 2002) was used as a positive control. Specificity was further verified by an antigen pre-absorption assay. By two rounds of IHC, the following 15 purified subclones were selected as highly promising Abs for the specificity and intensity of staining: 13-1, 13-3, 13-7, 13-8; 20-5, 20-7, 20-12, 20-6; 28-1, 28-8, 28-12; 29-8; 49-5, 49-7 and 49-9. Immune response data for these selected Abs are summarized in Table 5.

Table 5. Rabbit anti-hPD-L1 mAb immune response

* CHO cells stably transfected with PD-L1 versus CHO-S control;
^† PD-L1 positive tissues included placenta and one non-small cell lung cancer; detection to “very high” expression suggests better sensitivity in detecting PD-L1 of the membrane.

Additional assays were performed to further characterize purified Ab clones, including determining binding affinity and cross-competition in Abs by surface plasmon resonance (to identify overlapping vs. different epitope regions). Was. All Ab showed high binding affinity ^{_{(K D <10 -9 M)}} . These 15 purified clones were also analyzed by IHC in standard sections or FFPE low-density TMA for various cells and tissue types known to be positive or negative for cell surface expression of PD-L1. Rescreened. Rabbit IgG was used as an isotype control and mAb 5H1 was used as a positive control. At high concentrations (10 μg / ml), clones 28-x and 49-x showed low to moderate levels of background staining in tissue, while clone 13-x did not show background staining, and 13-x The clone was suggested to have a wide dynamic range. The 20-x clones showed varying degrees of background staining, which was mainly cytoplasmic and diffuse. Rabbit clone 28-8 (K _D = 100 pM as determined by SPR), a clone with very strong specific detection of PD-L1 on membrane, as the primary Ab for subsequent IHC assays chosen. MAb 28-1,28-12,20-12 and 29-8, respectively, had 130pM, 94pM, _{K D} values of 160pM and 1200PM. heavy chain variable _{(V H)} and light (kappa) chain variable mAb28-8 (V _κ) sequence of the region are described in each SEQ ID NO: 35 and 36. The 28-8 Ab was shown to recognize a different epitope than the mouse mAb 5H1, based on SPR analysis. Clones 28-1, 28-12, 29-8 and 20-12 were the next best Abs for strong detection of PD-L1 on membranes in FFPE tissues. mAb 13-1 had the best specificity for detection of PD-L1 in membranes, but the maximum detection level was lower than that of the other major Abs. Western blotting was also performed with plus / minus antigen competition to demonstrate the specificity of the top selected Abs for PD-L1.

  The binding of mAbs 5H1 and 28-8 to membrane PD-L1 was compared in FFPE test tissue samples containing tumor cells from different tumor types and tumor-infiltrating inflammatory cells. Membrane PD-L1 expression was assessed using the Histoscore method performed by two independent pathologists. Four NSCLC, two MEL, and two RCC tumors stained at 28-8 at 2 mg / ml and 5H1 at 5 mg / ml. The data is listed in Table 6 and is shown graphically in FIG. Rabbit mAb 28-8 shows better detection (higher histo-score) for 7 out of 10 samples using 2.5 times less Ab than only mAb 28-8 in one sample. Histo score for 5H1 was slightly higher.

Table 6. Comparison of mAbs 28-8 and 5H1 by Histo-score analysis

  Taube et al. (2012) demonstrated that mAb 5H1 bound to the cell surface by flow cytometry in cultured cells and demonstrated the specificity of PD-L1 binding to competitively bind 5H1 mAb to tissue sections. Confirmed using a blocking PD-L1 fusion protein. These authors also compared 5H1 previously described by Gadiot et al. (2011) to the rabbit polyclonal anti-hPD-L1 Ab, 4059, which showed a cell surface staining pattern in the FFPE sample, indicating pAb 4059. Demonstrated extensive cytoplasmic staining. Furthermore, when 40H1 was compared to pAb4059 by Western blot analysis, in contrast to 5H1, which specifically detected the 50 kDa band of glycosylated PD-L1, Ab4059 had the same expected mass of glycosylated PD-L1. In addition to the corresponding 50 kDa protein, it bound multiple proteins in lysates of melanoma cells (Taube et al., 2012). Contrary to the results of Taube et al. (2012) and the results described herein (summarized in Table 7), Gadiot et al. (2011) found that mAb 5H1 showed high levels of background staining in FFPE tissue samples. And found that pAb 4059 caused sufficient specific staining of PD-L1 in FFPE samples. None of the 13 other Abs tested by Gadiot et al. (2011) stained FFPE tissues that gave high background staining or were not blocked by PD-L1 fusion protein competition and PD- The difficulty in obtaining anti-PD-L1 Abs that specifically bind to L1 was highlighted.

  In this study, an automated IHC assay (see Example 10) was performed using several commercially available anti-PD-L1 Abs and 5H1 (Dong et al., Supra) on FFPE tissue samples containing various cells expressing PD-L1. 2002) was used to evaluate binding. The results summarized in Table 7 indicate that the commercially available Abs tested did not express PD-L1 or CHO cells expressing PD-L1 versus untransfected parent not expressing PD-L1. 4 shows that PD did not specifically recognize membrane PD-L1 expression in human tissues known to clearly distinguish CHO cells. The inability of polyclonal Ab (pAb) 4059 to bind specifically recognized membrane PD-L1 is consistent with the findings of Taube et al. (2012). The binding of 28-8 is similar to that of 5H1 in this assay, but histoscore analysis suggests that 28-8 works better than 5H1.

Table 7. Binding of mAb to FFPE sample containing PD-L1 expressing cells

* CHO cells stably transfected with PD-L1 versus parental CHO-S negative control;
^† PD-L1 positive tissue contained tonsils and / or thymus;
mAb, mouse monoclonal Ab; pAb, rabbit polyclonal Ab.

Example 10
Development of an automated IHC protocol to assess PD-L1 expression An automated IHC protocol was developed to assay PD-L1 expression in FFPE specimens. Tissue sections (4 μm) were mounted on slides and deparaffinized in an autostainer (Leica) by immersion twice in xylene for 5 minutes, 95% (v / v) EtOH twice in 100% EtOH for 2 minutes each. 2 times, 70% (v / v) 1 times EtOH, and rehydrated by soaking once in deionized water _(dH 2 O) to. Antigen retrieval was performed using an antigen stimulator (Biocare Medical Decloaking Chamber Plus) and Dako pH6 buffer, heated to 110 ° C. (P1) for 10 minutes, then the next step (P2 FAN ON at 98 ° C .; 90 (° C., FAN OFF). Slides were cooled at room temperature (RT) for 15 minutes and rinsed with water for about 1 minute.

Reagents were placed on an autostainer (BioGenex i6000) and tissue areas were defined using a pap pen. The IHC assay, which works using the autostainer in research mode, included the following steps: neutralize endogenous peroxidase using Peroxidase Block (Leica) for 10 minutes, and then with IHC wash buffer (Dako). Rinse 3 times; apply Protein Block (Leica) to slides, incubate for 10 min at RT, then wash 3 times with wash buffer; apply primary Ab (2 μg / ml) to slides, 1 hr at RT Incubate and then wash 6 times with wash buffer; add Post Primary Block (NovoLink Kit) to slides, incubate for 30 minutes, then wash 6 times with wash buffer; add NovoLink Polymer (NovoLink Kit) to slides Incubate for 30 minutes, then wash buffer In Wash 6 times; the DAB chromogen substrate (NovoLink Kit) was added, expanded 3 minutes, then 5 times rinsed with dH ₂ O at RT, counterstained with 1 minute hematoxylin at RT (NovoLink Kit), then, Wash 3 times with dH ₂ O for 5 times at RT. The primary Ab was selected from the rabbit anti-PD-L1 Ab shown in Table 4; mAb 28-8 was the preferred Ab. Rabbit IgG (Dako) was used as a negative control. Tissue sections are dehydrated using a Leica autostainer by washing once for 2 minutes in 70% EtOH, twice for 2 minutes in 95% EtOH, and 3 times for 2 minutes in 70% EtOH, and for 5 minutes in xylene. It was cleared by washing three times. Sections were mounted permanently on slides with permount, covered with coverslips, and transferred to a chemical hood for drying.

Example 11
Design of Phase 1 Clinical Trial of Anti-PD-L1 Antibody Trial Design Phase 1 trial was performed on BMS-936559 (12A4 in this specification and US Pat. No. 7,943,743) in patients with selected advanced solid tumors. (Also referred to as) to assess safety and tolerability. Secondary objectives included an initial assessment of the anti-tumor activity of BMS-936559 and a pharmacokinetic assessment. Pharmacokinetic measurements included preliminary objectives. Patients were treated in a 6-week cycle of BMS-936559 administered as a 60-minute intravenous infusion every two weeks on days 1, 15, and 29 of each cycle. Up to 16 cycles of treatment were continued until the patient experienced unacceptable toxicity, disease progression, or withdrew consent. In some patients that were clinically stable, treatment beyond the initial disease progression was allowed until further progression was confirmed.

Dose escalation Patients with advanced NSCLC, MEL, CRC, RCC, ovarian (OV), gastric (GC), breast (BC) and pancreatic (PC) carcinomas were eligible for enrollment. Safety was evaluated at doses of 0.3, 1, 3, and 10 mg / kg using an accelerated titration design. One patient who had ≧ grade 2 drug-related AE during cycle 1 was enrolled in each consecutive cohort. Next, two additional patients were enrolled at that dose level and the trial was transferred to a standard 3 + 3 design. Intra-patient dose escalation or tapering was not allowed. If less than one-third of the patients had dose-limiting toxicity, the maximum tolerated dose (MTD) was defined as the highest dose.

Cohort Expansion Initially, five expansion cohorts (n = 16 / cohort) were enrolled at 10 mg / kg for patients with NSCLC, MEL, RCC, OV and CRC. Based on the initial signal of activity, additional extended cohorts (up to n = 16 / cohort) were added to MEL (at 1.0 and 3.0 mg / kg), NSCLC (1, 3, or 10 mg / kg randomized flats). Epithelial or non-squamous epithelium tissue cohort), and PC, BC and GC at 10 mg / kg.

Patients Patients need to demonstrate advanced NSCLC, MEL, RCC, OV, CRC, PC, GC or BC and failed appropriate therapy for at least one previous tumor for advanced / metastatic disease (Except for PC or GC patients who may be treatment naive). Other inclusion criteria include: ≥18 years, life expectancy ≥12 weeks, ≤2 U.S. East Coast Joint Oncology Group general condition, measurable disease as defined by RECIST v1.0, and sufficient blood Physiology, liver and kidney function included. Patients with treated brain metastases were tolerated when stable for at least 8 weeks. The main exclusion criteria are a history of autoimmune diseases or other diseases requiring steroid or immunosuppressive drug treatment, previous treatment with Abs that regulate T cells (anti-PD-1, anti-PD-L1 and (Including anti-CTLA-4), HIV, or a history of active hepatitis B or C.

  In this continuation test, NSCLC (n = 75), MEL (n = 55), CRC (n = 18), RCC (n = 17), OV (n = 17), PC (n = 14), GC (n = 7) or BC (n = 4) were treated with BMS-936559 for 34 months. It contains safety data. Efficacy was characterized in 160 patients with evaluable responses. Baseline demographic characteristics of the entire patient population and of the population for which response could be assessed were very similar (Brahmer et al., 2012). Of the treated patients, 86% have received prior chemotherapy and 28% have received immunological or biological therapy. Previous treatments with tumor types included immunotherapy (56%) and B-RAF inhibitor (9%) in patients with MEL; chemotherapy with platinum (95%) and tyrosine kinase inhibitor (TKI) in patients with NSCLC. And 41%); and included nephrectomy (94%), anti-angiogenic therapy (82%) and immunotherapy (41%) in patients with RCC (for details on patient pretreatment, see Brahmer et al., 2012). reference).

Statistical analysis A total of 207 patients starting treatment on the day of analysis were used for baseline characteristics and AE summary. The efficacy population consisted of 160 patients whose response could be evaluated at least 7 months prior to the analysis date, estimating the response. The AE was coded using MedDRA v14.1. The individual best overall response was from a radiation scan measurement with a modified RECIST v1.0. OR was confirmed by at least one continuous tumor assessment. Further details on statistical methods are provided in Brahmer et al. (2012).

Example 12
Safety Assessment for Patients Treated with Anti-PD-L1 Antibody Safety assessments (laboratory and laboratory assessments) were performed at baseline and at regular intervals (cycle 1 period, weekly, and then biweekly). Performed in all treated patients. AE severity was graded based on NCI CTCAE, v3.0. Disease assessment via computer tomography (CT) scan or magnetic resonance imaging was performed at baseline and before each treatment cycle.

  MTD did not reach the highest tested dose of 10 mg / kg of BMS-936559. The median period of treatment was 12 weeks (range 2.0-111.1 weeks). A relative dose intensity of 90% ≧ was achieved in 86% of patients. Twelve (6%) of 207 patients discontinued treatment for BMS-936559-related AEs (see Brahmer et al., 2012 for details).

  Regardless of causality (any grade), AEs were reported in 188 of the 207 patients. BMS-936559-related AEs assessed by the investigator were observed in 126 of 207 (61%) patients. The most common drug-related AEs were fatigue, infusion reactions, diarrhea, joint pain, rash, vomiting, pruritus and headache. The majority of events were of low grade, and BMS-936559-related grade 3-4 events were observed in 19 of 207 (9%) patients (Brahmer et al., 2012). The range, frequency and severity of BMS-936559-related AEs were similar across dose levels, except for infusion reactions. Drug-related AEOSI with a possible immune-related etiology is observed in 81 of 207 (39%) patients, including rash, hypothyroidism, hepatitis, sarcoidosis, endophthalmitis, diabetes and myasthenia gravis Were included in each case alone (Brahmer et al., 2012). These AEs were predominantly grade 1-2 and were generally reversible upon treatment interruption or discontinuation. Strikingly, nine patients were treated with corticosteroids for the management of AEs. AEs were improved or resolved in all patients. In addition, four of these nine patients maintained disease control despite treatment with corticosteroids. Endocrine AEs were managed with replacement therapy and, at the discretion of the treating physician, the patient resumed treatment with BMS-936559. Infusion reactions were observed predominantly at 10 mg / kg in 21 out of 207 (10%) patients. The patients were grade 1-2 except for one grade 3 event at 10 mg / kg. Infusion reactions were generally rapidly reversible with antihistamines and antipyretics, in some cases corticosteroids. Antihistamines and antipyretic prophylaxis regimens were performed throughout the study. Patients with a grade 1-2 infusion response were able to continue treatment with BMS-936559 at a reduced infusion rate, with a prophylactic antihistamine and antipyretic. BMS-936559-related severe AEs occurred in 11 out of 207 (5%) patients. On the date of the data analysis, 45 patients (22%) died (Brahmer et al., 2012); no drug-related deaths were observed.

Example 13
Pharmacokinetic / Pharmacokinetic Analysis for Anti-PD-L1 Antibody For PK analysis, serial blood samples were collected and the serum concentration of BMS-936559 was quantified by ELISA. Peripheral blood mononuclear cells were isolated from patients at baseline and after one treatment cycle, and PD-L1 RO by BMS-936559 was assayed on circulating CD3-positive T-cells by flow cytometry (Brahmer et al., 2010).

  The serum concentration of BMS-936559 was dose-dependently increased from 1-10 mg / kg (n = 131). Areas under the geometric mean curve (0-14 days) for 1, 3, and 10 mg / kg dose levels were 2210, 7750 and 36620 μg / mL · hr, respectively (variable coefficient [CV] 34-59%); The geometric mean peak concentrations at these dose levels were 27, 83 and 272 μg / mL, respectively (CV 30-34%) (after the first dose). The half-life of BMS-936559 was estimated to be approximately 15 days from population pharmacokinetic data. PD-L1 RO in CD3-positive peripheral blood lymphocytes was evaluated in 29 MEL patients at the end of one cycle of treatment at a BMS-936559 dose of 1-10 mg / kg. Central RO was over 65% for all groups (Brahmer et al., 2012).

Example 14
Anti-tumor Efficacy Demonstrated by Anti-PD-L1 Antibody 160 of 207 treated patients could be assessed for response by February 2012, patients with NSCLC, MEL, CRC, RCC, OV and PC But did not include patients with GC or BC. Clinical activity was observed at all doses ≧ 1 mg / kg (Brahmer et al., 2012). OR (confirmed complete [CR] or partial [PR] response) was measured by patients with MEL, NSCLC, RCC and OV (FIGS. 10-13) as illustrated by typical spider plots and CT scans (FIGS. 10-13). Observed in Table 8), many ORs were also persistent (Table 9). Four additional patients had a sustained decrease in target lesions in the presence of new lesions, consistent with an "immune-related" pattern of response. However, these patients were not classified as responders for the purpose of calculating response rates. Anti-tumor responses and / or long-term stable disease (SD) have been observed in patients with various previous treatments. OR was observed even in patients with a broad burden of metastatic disease.

  In patients with MEL, there were 9 ORs with response rates of 6%, 29% and 19% at the 1, 3 and 10 mg / kg dose levels, respectively. Three MEL patients achieved CR. All 9 MEL patients who experienced OR started treatment> 1 year prior to data analysis; these 5 had a response period> 1 year. In addition, 14 MEL patients (27%) had SD lasting ≧ 24 weeks. In patients with NSCLC, there were 5 ORs with 8% and 16% response rates at the 3 and 10 mg / kg dose levels, respectively. There was OR in patients with non-squamous epithelium (n = 4) or squamous epithelium (n = 1). All five NSCLC responders started treatment ≧ 24 weeks prior to data analysis; three of these had responses lasting ≧ 24 weeks. Six additional NSCLC patients had SD lasting ≧ 24 weeks. At all 10 mg / kg doses, one of 17 patients with OV had one PR (6% response rate) and three patients (18%) with SD lasting ≧ 24 weeks. In patients with RCC, 2 of 12 (12%) patients treated with 10 mg / kg had an OR with a response lasting 4 and 18 months, respectively. Seven additional RCC patients had SD lasting ≧ 24 weeks.

Table 8. Clinical activity of BMS-936559 in 160 patients, evaluable response *

CI is confidence interval, MEL is melanoma, RCC is renal cell carcinoma, NSCLC is non-small cell lung cancer, OV is ovarian cancer, RCC is renal cell carcinoma, N / A is not applicable, ORR Indicates objective response rate (complete response + partial response), SD indicates stable disease, and PFSR indicates progression-free survival.
^* Efficacy population started treatment at least 7 months prior to the analysis date and had measurable disease with baseline tumor assessment and at least one of the following: tumor assessment, clinical progression or death at study Consists of patients whose response can be evaluated.
^{含 む} Including two CRs
^{含 む} Including one CR
^{§ The} objective response rate ({[CR + PR] {n} x 100) is based on responses identified only with confidence intervals calculated using the Cropper-Pearson method.
** Progression-free survival was calculated by Kaplan-Meier methodology with confidence intervals using the Greenwood method and was the percentage of patients who did not progress and were alive at 24 weeks.

  Individual patient responses were determined by RECIST v1.0 with modifications (see Brahmer et al. (2012) Test Protocol in N Engl J Med (submitted) for more information).

Table 9. Duration of objective response to BMS-936559 *

* MEL indicates melanoma, NSCLC indicates non-small cell lung cancer, RCC indicates renal cell carcinoma, and OV indicates ovarian cancer.
^時間 Time to first confirmed response, time to confirmed progress, death, or for censored data (indicated by “+”), to last tumor assessment.

Example 15
Design of a Phase 1 Clinical Trial of Anti-PD-1 and Anti-CTLA-4 in Advanced MEL In a Phase 1 study, a continuous cohort of patients was treated with increasing doses of nivolumab and ipilimumab administered simultaneously intravenously. Treated (concurrent regimen), and separately, two cohorts of patients were previously treated with ipilimumab and received nivolumab alone (sequential regimen).

  In the concurrent regimen, patients received nivolumab and ipilimumab four times every three weeks during the induction period and then four times nivolumab alone every three weeks. Next, the combination treatment was continued up to 8 times every 12 weeks during the maintenance period. Nivolumab was administered first when both drugs were administered together. Within the cohort, nivolumab and ipilimumab doses were kept constant during induction and maintenance. The dose limiting toxicity evaluation period spanned 9 weeks. Tumor assessments were at weeks 12, 18, 24 and 36, and every 12 weeks thereafter.

  In a continuous regimen, patients previously treated with ipilimumab before study enrollment received up to 48 nivolumab every two weeks. Nivolumab treatment started within 4-12 weeks after ipilimumab monotherapy. Tumor assessment was at 8 weeks and every 8 weeks thereafter. Tumor response was determined using mWHO and immune-related mWHO criteria in both regimens.

  After completion of treatment, patients without confirmed disease progression were followed for up to 2.5 years. Patients with disease progression at CR, PR or SD> 24 weeks and later can be retreated with the original regimen. Safety assessments were performed for each protocol. AE severity was graded according to the National Cancer Institute Adverse Events Common Terms Standard, version 3.0. Disease assessment was performed on a protocol-by-protocol basis, as appropriate, using CT and / or MRI.

Dose Escalation and Cohort Expansion A study is first designed to evaluate a co-regulation using a standard 3 + 3 design for the dose escalation phase, then a cohort expansion for a total of up to 16 patients at maximum tolerated dose or maximum dose. did. The dose limiting toxicity (DLT) evaluation period for dose escalation was 9 weeks. No intra-patient dose escalation was observed and patients who experienced DLT discontinued treatment. Patients who withdrew from the study during the DLT assessment period for reasons other than drug-related toxicity could be replaced. The protocol was modified to allow N = up to 12 patients to extend any concurrent regimen cohort during dose escalation. Two consecutive regimen cohorts (6 to 16 patients each) were later added; patients were treated with nivolumab (1 mg / kg or 3 mg / kg) after receiving prior ipilimumab.

Patients Eligible patients are 18 years of age and have a measurable diagnosis of unresectable stage III or IV melanoma; 0-1 general condition of the US East Coast Cooperative Oncology Group, where 0 is asymptomatic and There was one mildly symptomatic; sufficient organ function; and life expectancy> 4 months. Active untreated central nervous system metastases; history of autoimmune disease; previous treatment with antibodies that modulate T cells (except ipilimumab for continuous regimen cohort); HIV; or patients with hepatitis B or C Was removed.

  In a continuous regimen cohort, patients were required to receive three prior doses of ipilimumab with the last dose administered within 4-12 weeks of nivolumab initiation. Patients with a history of CR, progression with evidence of clinical deterioration, or a high grade AE associated with ipilimumab were excluded (see Wolchok et al. 2013a, for details of the protocol).

  86 patients were treated from December 2009 to February 2013, 53 were on a concurrent regimen and 33 were on a continuous regimen. Baseline patient characteristics are described in detail in Wolchok et al. (2013a). In a concurrent and sequential regimen, 38% and 100%, respectively, of patients received prior to systemic therapy. The majority of patients had M1c disease and> 30% had elevated serum lactate dehydrogenase (LDH). Many patients enrolled in the continuous regimen cohort demonstrated radiation progression (73%) with prior ipilimumab treatment.

PD-L1 immunohistochemistry Pre-treated PD-L1 expression was measured by rabbit anti-PD-L1 mAb, IHC in FFPE tumor specimens using 28-8, and an automated assay developed by Dako (Carpinteria, CA). . Ab specificity was assessed by Western blotting on recombinant PD-L1 protein and lysates from cell lines expressing and not expressing PD-L1. Evaluation of staining patterns in normal human tissues and IHC assays with and without antigen competition were performed. The assay sensitivity, specificity, reproducibility, reproducibility and robustness of the immunohistochemical assay were tested and met all pre-specified acceptance criteria. Two pathologists whose treatment results were blinded independently read all clinical specimens and scored. A sample was defined as PD-L1-positive when 5% of the tumor cells showed any intensity of membrane PD-L1 staining in sections with 100 evaluable cells.

Statistical analysis All treated patients February 2013 (N = 86) were used to show analysis of baseline characteristics, safety, and absolute lymphocyte count (ALC), and PD-L1 staining. . The efficacy population received at least one dose of study treatment, had a measurable disease at baseline, and had at least one of:> 1 tumor assessment at treatment, clinical progression or It consisted of 82 patients with evaluable responses with death before tumor evaluation. The AE was coded using MedDRA, version 15.1. AEs selected for possible immunological etiology were identified using a previously defined list of MedDRA terms. The best overall response was obtained programmatically from tumor measurements provided by radiologists and investigators at the study site by modified WHO (mWHO) or immune-related response criteria (Wolchok et al. 2009). Total and partial responses were confirmed by at least one subsequent tumor assessment. Analysis was also performed to assess the magnitude of target lesion reduction by radiological assessment. Responses were characterized as intensity when they showed a 80% decrease from baseline baseline. Unconfirmed responses at the date of this analysis were also included in the estimate of total clinical activity.

Example 16
Safety assessment for MEL patients treated with anti-PD-1 and anti-CTLA-4 In a concurrent regimen (n = 53), AEs of all grades were observed in 98% of patients, regardless of attributes. Treatment-related AEs were observed in 93% of the patients, the most common being rash (55%), pruritus (47%), fatigue (38%) and diarrhea (34%). Grade 3-4 AEs, regardless of attributes, were observed in 72% of patients, whereas grade 3-4 treatment-related events were seen in 53%, the most common being lipase (13%), There was an increase in aspartate aminotransferase (13%) and alanine aminotransferase (11%). Six out of 28 (21%) patients had grade 3-4 dose limiting treatment-related events. Treatment-related severe AEs were reported in 49% of patients. General grade 3-4 treatment-related curated AEs included liver (15%), gastrointestinal (9%) and kidney (6%) events. Extreme cases of pneumonia and uveitis were seen, consistent with historical monotherapy experience. Eleven (21%) patients were discontinued due to treatment-related AEs.

  Cohort 3 (3 mg / kg nivolumab + 3 mg / kg ipilimumab) exceeded the MTD (3 out of 6 patients experienced asymptomatic grade 3-4 elevated lipase that lasted 3 weeks). Cohort 2 (1 mg / kg nivolumab + 3 mg / kg ipilimumab) was identified as MTD (grade 3 uveitis, grade 3 elevated AST / ALT in one patient each).

  In a continuous regimen (n = 33), all grades of AE were observed in 29 (88%) patients, regardless of attribute. Treatment-related AEs were observed in 24 (73%) patients, the most common of which included pruritus (18%) and lipase elevation (12%). Grade 3-4 AEs were observed in 11 (33%) patients, regardless of attributes, while grade 3-4 treatment-related AEs were observed in 6 (18%) patients and were the most common. A significant event was lipase elevation (6%). Treatment-related severe AEs were reported in 7 (21%) patients. Grade 3-4 endocrine events were indicated as treatment-related curative AEs in two patients. One patient had grade 2 pneumonia. Three (9%) patients were discontinued due to treatment-related AEs.

  In both simultaneous and sequential regimens, treatment-related AEs can be managed with immunosuppressants and / or replacement therapies (for endocrine disorders) by generally established algorithms for ipilimumab, and are generally reversible. (See YERVOY® package insert). Of the 86 patients treated in the study, 28 of 38 (38%) with drug-related adverse events required management on systemic glucocorticoids. Three patients required additional immunosuppressive therapy with infliximab (2 patients) or mycophenolate mofetil (1 patient). No treatment-related deaths were reported. Further details of AEs and their management are provided in (Wolchok et al. 2013a).

Example 17
Efficacy demonstrated by combination of anti-PD-1 and anti-CTLA-4 in MEL patients Clinical activity was observed in both simultaneous and sequential regimens (Tables 10 and 11). In the concurrent regimen cohort, confirmed objective responses (OR) by mWHO criteria were observed across all doses in 21 of 52 patients (40%; 95% CI: 27-55) with evaluable responses. Was done. After noting some patients who have demonstrated a major response (approaching CR), have a selected empirical threshold of at least 80% tumor reduction to indicate the level of tumor regression that approaches complete response The number of patients was evaluated. This depth of response was not common in published studies of checkpoint blockade (Hodi et al., 2010; Topalian et al., 2012b). Sixteen patients had ≧ 80% tumor reduction at 12 weeks and included 5 CRs (Table 10, FIGS. 14A and 15-17). In addition to 21 patients with OR by mWHO criteria, 4 patients experienced objective responses according to immune-related response criteria, and 2 patients had unconfirmed responses. These patients were not included in ORR calculations. In the concurrent regimen, comprehensive evidence of clinical activity (conventional, unconfirmed or immune-related response or SD> 24 weeks) is observed in 65% of patients (95% CI: 51-78; Table 10). Was done. The large effect of the simultaneous combination is best understood in the waterfall plot (FIG. 14B). Responses were sustained in 19 of 21 responders over a period of weeks ranging from 6.1+ to 72.1+ at the time of data analysis (Table 12). In patients treated with MTD (Cohort 2, 1 mg / kg nivolumab + 3 mg / kg ipilimumab), OR occurred in 9 of 17 (53%; 95% CI: 28-77) patients and 3 CRs Was included. All nine responders achieved ≧ 80% tumor reduction at the originally scheduled treatment assessment (Table 10 and FIG. 14A).

  Of the patients in the continuous regimen cohort, 6 out of 30 patients achieved an OR with one CR (20%; 95% CI: 8-39). Four (13%) patients achieved 80% tumor reduction at 8 weeks (Table 11 and FIG. 18). Additional patients had immune-related responses (n = 3) or unidentified responses (n = 3). When objective, immune-related or unconfirmed responses or SD> 24 weeks were considered, evidence of clinical activity in a continuous regimen was observed in 43% (95% CI: 26-63). The waterfall plot shows that patients who did not respond prior to ipilimumab can respond to nivolumab later (FIG. 18C).

Table 10. Clinical activity in patients receiving a concurrent regimen of nivolumab and ipilimumab *

［（ＣＲ＋ＰＲ＋ｕＣＲ＋ｕＰＲ＋ｉｒＰＲ＋ＳＤ≧２４週＋ｉｒＳＤ≧２４週／応答を評価可能な患者数］×１００。
＊＊コホート２において２人のさらなる患者が、１２週後に行われた最初に予定された評価で≧８０％腫瘍減少をなし遂げた。 * CR indicates a complete response, PR indicates a partial response, uPR indicates an unconfirmed partial response, irPR indicates an immune-related partial response, SD indicates a stable disease, and irSD indicates an immune-related stable disease.
^患者 Patients whose response can be assessed receive at least one dose of study treatment, have a disease that can be measured at baseline, and one of the following: 1) tumor assessment at least one treatment, 2) clinical Patients with advanced or 3) death before tumor assessment at the time of the first treatment.
^患者 Patients who had PR after one tumor assessment but did not have enough follow-up time for confirmation of the first PR.
^{§Patients with} reduced target tumor foci in the presence of new lesions, consistent with immune-related PR or SD.
^¶ [(CR + PR) / number of patients whose response can be evaluated] × 100. Confidence intervals were estimated by the Cropper-Pearson method.

[(CR + PR + uCR + uPR + irPR + SD ≧ 24 weeks + irSD ≧ 24 weeks / number of patients evaluable for response] × 100.
** Two additional patients in Cohort 2 achieved ≧ 80% tumor reduction at the first scheduled assessment performed after 12 weeks.

Table 11. Clinical activity in patients receiving a continuous regimen of nivolumab and ipilimumab *

* CR indicates a complete response, PR indicates a partial response, uPR indicates an unconfirmed partial response, irPR indicates an immune-related partial response, SD indicates a stable disease, and irSD indicates an immune-related stable disease.
^患者 Patients whose response can be assessed receive at least one dose of study treatment, have a disease that can be measured at baseline, and one of the following: 1) tumor assessment at least one treatment, 2) clinical Patients with advanced or 3) death before tumor assessment at the time of the first treatment.
^患者 Patients who had PR after one tumor assessment but did not have enough follow-up time for confirmation of the first PR.

Table 12. Duration of confirmed objective response of individual patient

* Not available; no objective response confirmed at the time of this analysis.

Assessment of tumor PD-L1 expression and absolute lymphocyte count Changes in tumor PD-L1 expression and peripheral blood ALC were studied with biomarkers for nivolumab and ipilimumab monotherapy, respectively (Topalian et al., 2012b; Berman et al.). , 2009; Ku et al., 2010; Postow et al., 2012; Delyon et al., 2013). Tumor PD-L1 expression was characterized via IHC staining and pharmacokinetic changes in peripheral blood ALC were analyzed. Using a ≧ 5% cutoff to define PD-L1 positivity, tumor specimens from 21 of 56 (38%) patients were PD-L1-positive. OR was seen in patients with either PD-L1-positive (6/13) or PD-L1-negative (9/22) tumors among patients treated with the concurrent regimen (post-hocP values>0.99;Fisher's exact test). In a continuous regimen cohort, a numerically higher number of overall responses was observed in patients with PD-L1-positive tumor samples (4/8) compared to patients with PD-L1-negative tumors (1/13). Seen, but few.

  In contrast to observations with ipilimumab monotherapy, no consistent increase in ALC from baseline was detected in patients treated with co-combination or patients treated with nivolumab after ipilimumab treatment. In the concurrent regimen cohort, patients with low ALC at 5 to 7 weeks (<1000 cells / μL) (Ku et al., 2010) were similar compared to patients with normal ALC at 5 to 7 weeks (40%) OR (43%). Similarly, in the continuous regimen cohort, 17% of patients with low ALC had OR and 23% of patients with normal or high ALC had OR.

Sequence Listing Summary

References

Claims (17)

CDRl, CDR2 and CDR3 regions in the heavy chain variable region comprising a contiguously linked amino acid having the sequence set forth in SEQ ID NO: 35, and a continuous link having the sequence set forth in SEQ ID NO: 36 A monoclonal antibody or an antigen-binding portion thereof, which binds to a PD-L1 polypeptide, comprising a CDR1, CDR2 and CDR3 region in a light chain variable region containing the selected amino acids. The antibody or antigen-binding portion thereof has a heavy chain variable region comprising a continuously linked amino acid having the sequence set forth in SEQ ID NO: 35, and a continuous chain having the sequence set forth in SEQ ID NO: 36. 2. The monoclonal antibody or antigen binding portion thereof of claim 1, comprising a light chain variable region comprising linked amino acids. (I) the antibody of claim 1 or 2 or an antigen-binding portion thereof, and (ii) instructions for detecting PD-L1 expression in a tissue sample obtained from a subject in need of cancer treatment. kit. Antibodies that specifically bind to human programmed death 1 ("PD-1") or human programmed death ligand 1 ("PD-L1") ("anti-PD-1 antibody" or "anti-PD-L1 antibody") 4. The kit of claim 3, further comprising an antigen binding portion thereof, and instructions for administering the anti-PD-1 antibody to a subject identified as having positive PD-1 expression. The kit according to claim 3 or 4, wherein the tissue sample comprises tumor cells. An antibody or antigen-binding portion thereof according to claim 1 or 2 for identifying a subject that is suitable for immunotherapy comprising measuring PD-L1 expression in a test tissue sample obtained from the subject. The antibody or antigen-binding portion thereof, wherein the subject has cancer. 7. The antibody or antigen-binding portion thereof according to claim 6, wherein the subject having PD-L1 expression is identified as suitable for immunotherapy. 8. The antibody or antigen-binding thereof of claim 6 or 7, wherein the subject identified as suitable for immunotherapy is administered to the subject a composition comprising a therapeutically effective amount of an anti-PD-1 antibody. part. 9. The antibody or antigen-binding portion thereof according to any of claims 6 to 8, wherein PD-L1 expression is detected by an immunohistochemical assay of a test tissue sample. 10. The antibody or antigen-binding portion thereof according to claim 9, wherein the immunohistochemical assay is an automated immunohistochemical assay. Cancer is multiple myeloma, B cell lymphoma, Hodgkin lymphoma / primary mediastinal B cell lymphoma, non-Hodgkin lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, follicular lymphoma , Diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma The antibody or antigen-binding portion thereof according to any one of claims 6 to 10, which is a hematological malignancy selected from T-cell lymphoma, and T-lymphoblastic lymphoma. The antibody or antigen-binding portion thereof according to any one of claims 6 to 11, wherein the cancer is advanced, recurrent, metastatic and / or refractory cancer. 13. The antibody or antigen-binding portion thereof according to any of claims 6 to 12, wherein the cancer is melanoma, renal cell carcinoma, NSCLC, castration-resistant prostate cancer (CRPC), or colorectal cancer (CRC). 14. The antibody or antigen-binding portion thereof according to any of claims 6 to 13, wherein the test tissue sample is a formalin fixed paraffin embedded (FFPE) tissue sample. 15. The antibody or antigen-binding portion thereof according to any of claims 6 to 14, wherein at least 1% of the tumor cells express cell surface PD-L1. 16. The antibody or antigen-binding portion thereof according to any of claims 6 to 15, wherein at least 5% of the tumor cells express cell surface PD-L1. A method for measuring PD-L1 of a membrane on an isolated tumor cell, comprising performing an immunohistochemical assay using the antibody or the antigen-binding portion thereof according to claim 1 or 2.

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